Nucleic Acid Probe and Method of Detecting Genomic Fragments

ABSTRACT

Provided herein, among other things, is a method of processing a nucleic acid sample. In some embodiments, the method comprises a) hybridizing a sample comprising a target fragment to a nucleic acid probe comprising: i. a head sequence and a tail sequence, wherein the head and tail sequences are at the ends of a first oligonucleotide molecule; and ii. a splint sequence comprising, in order: an upstream flanking sequence that is complementary to the head sequence; a target complementary sequence that is complementary to the target fragment; and a downstream flanking sequence that is complementary to the tail sequence; thereby producing a hybridization product in which the ends of the target fragment are ligatably adjacent to the ends of the head and tail sequences in the first oligonucleotide molecule; and b) ligating the ends of the target fragment to the ends of the head and tail sequences of the first oligonucleotide molecule, thereby producing a cyclic product that comprises the target fragment and the head and tail sequences. Probes and kits for performing the method are also provided.

CROSS-REFERENCING

This application claims the benefit of UK Application No: 1321191.7,filed on Dec. 2, 2013, which application is incorporated by referenceherein.

FIELD OF THE INVENTION

The present disclosure relates to probes for detecting specific nucleicacid sequences in biological samples, especially probes for use inmultiplex methods of detecting multiple specific sequences in parallel,and to methods in which such probes are used for detecting fragments ofnucleic acid. In particular, the present disclosure relates to targetingDNA fragments from specific chromosomes for downstream analysis.

BACKGROUND

The human haploid genome contains 3 billion base pairs packaged in 23chromosomes, and the diploid genome has 6 billion base pairs in 23 pairsof chromosomes. The rapidity and convenience of modern sequencingtechnology enables many diagnostic questions to be approached usinghigh-throughput sequencing of an individual's entire genome or of thefull quantity of DNA in a sample. However, for many DNA diagnosticsapplications, it is only necessary to investigate a subset of thegenome, focussing on the region or regions known to be associated withthe particular disorders under investigation.

A number of techniques have been described for reducing the complexityof the genome before analysis. Where only a single, short region of thegenome is required to be analysed, this may be done usingstraightforward PCR to amplify the sequence using primers to knownregions on either side. However, when it is desired to amplify manyregions of a genomic sample for analysis, amplification artefacts canarise as a result of performing multiple different amplificationstogether in the same reaction mixture.

WO2003/044216 (Parallele Bioscience, Inc.) and US20090004701A1 (MalekFaham) described a method of multiplex amplification of target nucleicacids, in which common oligonucleotide primers were ligated to sitesinternal to single-stranded nucleic acid fragments. The common primingsites were appended to each of a plurality of different target sequencesto allow their stoichiometric amplification.

WO2005/111236 (Olink AB) also described a method of identifyingsequences in the human genome by amplifying specific target sequences.The method involved fragmenting the genomic sample into fragments havingat least one defined end sequence. Selector constructs, all comprising aprimer pair motif, were brought in contact with the fragments. Afterligation, the selected target sequences were amplified in parallel usinga primer-pair specific for the primer-pair motif common to theselectors. The selector constructs described in WO2005/111236 had a longoligonucleotide hybridised to a short oligonucleotide, each selectorconstruct having one or two protruding ends complementary to a definedend sequence of a fragment containing the target sequence. Contactingthe selectors with the target fragments resulted in hybridisation of thetarget fragment between protruding ends of the selector or selectors. Inthe case of a single selector with two protruding ends thishybridisation produced a circularised construct. In the case of a pairof selectors each with one protruding end this formed a linearconstruct. Ligation and sequencing of the selector constructs containingthe target fragments allowed the target sequence to be determined. Sincethe selector constructs hybridise only to the end portions of thefragment containing the target sequence (or to one end portion and oneinternal portion), the method allowed selection of target sequences thatdiffered in the non-hybridising portions, so that each selector moleculecould hybridise to a variety of different target sequences. The identityof the exact target was then determined by amplifying and sequencing theconstructs. WO2005/111236 proposed using the selectors in methods ofanalysing genetic variability or for DNA copy number measurements.

GB2492042 described a variation of the selector method, in which thefragments were contacted with a partially double-stranded probecomprising a selector oligonucleotide and at least one vectoroligonucleotide. The selector oligonucleotide contained two non-adjacentregions specific for the target fragment and a non-target specificregion which comprised at least two binding sites for the vectoroligonucleotide. The vector oligonucleotide was not complementary to thetarget sequence, and included a nucleotide sequence complementary to thevector-binding site on the selector oligonucleotide. The vectoroligonucleotide also contained elements for detection/enrichment. In themethod, complementary portions of the probe oligonucleotides werehybridised to the target fragment, followed by ligating the vectoroligonucleotide(s) and target to produce a probe-target fragment hybrid,which was then detected.

A development of the selector technology was described in WO2011/009941(Olink Genomics AB), describing ligation of one end of a fragment ofdigested genomic DNA to a probe. Compared with the earlier selectorprobes, which involved binding to two regions of the target fragment andwhere the sequence to be isolated was typically bounded by two regionsof known sequence, the probes in WO2011/009941 were described for usewhere there was only one known region of sequence. Some embodiments ofthe probes in WO2011/009941 contained elements for immobilisation to asolid phase. Ligation of the target nucleic acid fragment to the proberesulted in a stable capture of the target fragment and allowed the useof highly stringent washing steps to remove non-ligated fragments,resulting in a high specificity.

Also known are padlock probes. Padlock probes are linearoligonucleotides with target complementary sequences at the ends and anon-target complementary sequence in between. When hybridised to thecorrect target DNA sequence, the two ends of the probe are broughttogether head to tail and can be joined by DNA ligase. Ligation isinhibited by mismatches at the ligation junction, so successful ligationof the padlock probe depends on highly specific hybridisation to thetarget sequence, allowing the probe to distinguish between highlysimilar target sequences and selectively padlock its exact target. As aconsequence of the helical nature of double stranded DNA, thecircularised probe molecule is catenated to the target DNA strand.

It was known to amplify the circularised padlock probes using rollingcircle replication, also known as rolling circle amplification. Rollingcircle replication was described in U.S. Pat. No. 5,854,033 (Lizardi).Rolling circle replication is an amplification of a circular nucleicacid molecule using a strand displacing DNA polymerase, resulting inlarge DNA molecules containing tandem repeats of the amplified sequence.The DNA polymerase catalyses primer extension and strand displacement ina processive rolling circle polymerisation reaction that proceeds aslong as desired. It results in an amplification of the circularisedprobe sequence orders of magnitude higher than a single cycle of PCRreplication and other amplification techniques in which each cycle islimited to a doubling of the number of copies of a target sequence.Additional amplification can be obtained using a cascade of stranddisplacement reactions.

Fredriksson et al. (Nucleic Acids Res. 35(7):e47 2007) described“Gene-Collector”, a method for multiplex amplification of nucleic acidsusing collector probes which contain adjacent sequences complementary tothe cognate primer end sequences of desired PCR products, so thatbinding of the collector probes to the PCR products brings the ends ofthe PCR products together to form a DNA circle. Universal amplificationis then performed using rolling circle amplification to generate a finalproduct of concatamers of target sequences. This method allows thecorrect amplicons in a multiplex PCR reaction to be selectivelydetected, because the end sequences of the correct amplicons are acognate primer pair and are circularised by the collector probe, whereasPCR artefacts combining a primer from one pair with a primer fromanother pair are not circularised.

SUMMARY OF THE INVENTION

The present disclosure provides improved methods and probes foranalysing nucleic acid fragments, such as fragmented genomic DNA. Someembodiments of the invention relate to probes and to their use inmethods of testing samples for the presence of a target single strandednucleic acid fragments. Some embodiments of the invention relate toprobes which comprise

a targeting oligonucleotide containing a target-complementary sequencewhich is the complement of the target fragment and a flanking sequenceadjacent to the target-complementary sequence, and

an oligonucleotide sequence having a free 5′ or 3′ end,

wherein hybridisation between the fragment and the probe templates thetarget fragment for ligation to the free 5′ or 3′ end of theoligonucleotide sequence.

Some embodiments of the invention further relate to probes whichhybridise along the length of a single stranded nucleic acid fragmentand ligate to each end of the fragment. Such probes comprise anoligonucleotide sequence having a free 5′ end and an oligonucleotidesequence having a free 3′ end, for ligation to each end of the targetfragment. The ligation product is then detected, allowing a highlyspecific targeting and detection of the defined nucleic acid fragments.

A method according to some embodiments of the invention may comprisedigesting DNA to fragments with defined sequence, denaturing theresulting DNA fragments to single stranded fragments (targets) andmixing the targets with probes as described herein. Hybridisation of thetargets to the probes produces templates for ligation to specificallyconnect the target to a corresponding probe to generate either a circleor a linear ligation product. The ligation products may then beenriched, for example by exonucleases or solid-phase chemistry, andoptionally amplified by rolling circle amplification, PCR, or other DNAamplification methods.

A key advantage of some embodiments of the present invention lies in theanalysis of a multitude of DNA fragments in parallel. A multitude of DNAfragments may be specifically targeted and selected for downstreamanalysis. This is particularly useful for the non-invasive prenataltesting (NIPT) of cell-free foetal DNA in the maternal bloodstream,where counting of thousands of chromosome-specific DNA fragmentsproduces a very precise quantification.

In one aspect, a method of testing a sample for the presence of a targetnucleic acid is provided. The method typically involves generatingdefined target nucleic acid fragments, contacting the sample with aprobe that hybridises along the length of the target fragment andprovides ligatable junctions in both the 3′ and 5′ end of the fragment,ligating the target fragment to the probe at both the 3′ and 5′ end, andthen detecting the new nucleic acid molecule formed by the doubleligation event.

One aspect provides a method of testing a sample for the presence of atarget nucleic acid, comprising:

(i) providing a sample of fragmented nucleic acid(ii) providing denaturing conditions under which the target fragment issingle stranded(iii) contacting the sample with a nucleic acid probe comprising

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and the target fragment,if present, hybridises to the target-complementary sequence, therebypositioning the ends of the target fragment in juxtaposition with the 5′end of the head sequence and the 3′ end of the tail sequence(v) providing conditions for ligation so that, if the target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment, and(vi) detecting whether the product of double ligation is present,

wherein detecting the product of double ligation indicates the presenceof the target fragment in the sample.

In contrast with most other DNA selection and detection approaches, thepresent method may be particularly useful when the entire nucleic acidfragment is pre-defined or pre-determined—that is, when the sequence ofthe target fragment is known in advance. In some implementations of thepresent method, the target fragment is the product of a specificfragmentation of nucleic acid, rather than a random fragmentation suchas may be produced by physical means such as shearing or sonication.Specific fragmentation of nucleic acid may be achieved using restrictionenzymes, PCR, or other sequence directed fragment end definition.

It is desirable for the targeting oligonucleotide to contact the entiretarget fragment, to ensure specific binding of the precise targetsequence. This contrasts with earlier approaches where probes weredesigned to hybridise with an end or ends of the fragment and/or to aninternal region but not to bind along the length of the target fragment.Indeed, limited binding to the target fragment was a deliberate designfeature in many earlier probes, since it allowed fragments to betargeted and detected when their sequence was only partly known. Byspecifically targeting fragments of known sequence—subject to thepossibility of slight sequence variability resulting from differentalleles in a population, where applicable—the probes and methods of thepresent method may allow precise binding and detection of the desiredtarget fragment with very low risk of false-positive results.

The double ligation of the target fragment further may contribute to thehigh specificity of the method. The probe becomes ligated to the targetsequence at each end, i.e., to the 5′ and 3′ ends of the single strandedfragment of nucleic acid. Thus, the ends of the target which werespecifically generated by fragmentation may be specifically detected bysequence-specific ligation to the head and tail sequences. Thesequence-specific nature of the ligation is achieved through therequirement for hybridisation of both the target fragment and the headand tail sequences to the targeting oligonucleotide, and through thesensitivity of DNA ligase which is inhibited by base pair mismatches.Hybridisation of the target fragment to the targeting oligonucleotidecontributes to the specificity of the binding but, in contrast with theligation reactions which provides highest selectivity with respect tomismatches at the 3′ and 5′ ends of the target fragment, thehybridisation is destabilised the most by internal mismatches in thecentral part of the target.

The targeting oligonucleotide acts to template ligation of the targetfragment to the head and tail sequences. The head and tail sequenceshybridise to the flanking sequences, defining a gap between the 5′ endof the head sequence and the 3′ end of the tail sequence. The targetfragment hybridises to the target-complementary sequence in the gap,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tailsequences. Preferably, the annealing of the target fragment and the headand tail sequences to the probe generates two perfectly matchedligatable junctions. The product of double ligation is then a continuousstrand of nucleic acid comprising the head and tail sequences and thetarget fragment.

A number of possible designs of the probe are contemplated. For example,the 5′ and 3′ ends for ligation to the target fragment may be providedby head and tail sequences on two separate backbone oligonucleotides, orby head and tail sequences at respective ends of a single backboneoligonucleotide which loops to position the target fragment between the5′ and 3′ ends.

In the first case (two separate backbone oligonucleotides), ligation ofthe target fragment to the two backbone oligonucleotides produces alinear strand of nucleic acid comprising the target fragment between thehead and tail sequences.

In the second case (single looped backbone oligonucleotide), ligation ofthe target fragment produces a circle of nucleic acid comprising thetarget sequence between the head and tail sequences.

In further versions, one or both of the head and tail sequences may beprovided on the targeting oligonucleotide itself, so that the targetingoligonucleotide forms a looped structure under annealing conditions.Depending on the design, the product of double ligation in such casesmay either be a linear or a circular nucleic acid molecule.

Detection of the product is dependent on successful ligation of thetarget fragment to the head and tail sequences to form a continuousstrand of nucleic acid. In general, the product of double ligation isdetected using an approach that requires both ligation events to occurin order to generate a signal. For example, detection may compriseamplification across both ligation junctions (e.g., by PCR or, forcircularising embodiments of the probe, rolling circle replication), orcapturing the continuous nucleic acid strand at one end and detectingits other end. The covalent attachment of the target fragment to theprobe by ligation forms a strong bond, so stringent washing may be usedto remove non-ligated nucleic acids in which the head and tail sequencesare not covalently attached, their mutual hybridisation to the targetingoligonucleotide being disrupted by the washing.

These features of the method and the probe enable a highly specificselection of target fragments. When the methods are applied formultiplex detection of a plurality of target fragments in parallel, avery precise detection and quantification of the target nucleic acid ispossible. As a result of its high specificity, the present method may beespecially suitable for diagnostic use in small samples and/or fordetecting very small differences in relative amounts of different targetnucleic acids, for example in diagnosing aneuploidies in foetalchromosomes from a sample of maternal blood or in detecting the presenceof trace amounts of tumour DNA in a sample of normal tissue from apatient or detection of nucleic acid fragments from infectious agents.

Having a highly specific target fragment recognition enables use of arelatively high probe concentration without generating false positivesignals, thereby increasing the yield and efficiency of the reaction.This may be of high importance in diagnostic applications where lowvariability is important and targets may be present in low numbers forexample in NIPT by analysis of cell free DNA, detection of cell freecirculating cancer DNA and detection of DNA from infectious agents. Someembodiments of the present method enables highly specific analysis ofshort DNA fragments, which is of importance in applications for analysisof fragmented DNA like cell free DNA in blood, or formalin fixedparaffin embedded DNA.

With reference to FIGS. 3 and 4, provided herein, among other things, isa method of processing a nucleic acid sample. In some embodiments, themethod may comprise: a) hybridizing a sample (e.g., a sample that hasbeen digested with a restriction enzyme) comprising a target fragment (a“DNA target”) to a nucleic acid probe comprising: i. a head sequence anda tail sequence, wherein the head and tail sequences are at the ends ofa first oligonucleotide molecule; and ii. a splint sequence (where theterm “splint sequence” is intended to refer to a sequence in anoligonucleotide that, when hybridized to two or more otherpolynucleotides, acts as a “splint” to position the polynucleotides nextto one another so that they can be ligated together, as illustrated inFIGS. 3 and 4). As shown in FIGS. 3 and 4, the splint sequence (which isreferred to as a “targeting oligonucleotide” in some cases) used in thismethod contains an upstream flanking sequence that is complementary tothe head sequence; a target complementary sequence that is complementaryto the target fragment; and a downstream flanking sequence that iscomplementary to the tail sequence. This hybridization step produces ahybridization product in which the ends of the target fragment areligatably adjacent to the ends of the head and tail sequences in thefirst oligonucleotide molecule, where the term “ligatably adjacent” inthe context of two sequences that are ligatably adjacent to one another,means that there are no intervening nucleotides between twooligonucleotides and they can be ligated to one another using a ligase.The next step of the method comprises b) ligating the ends of the targetfragment to the ends of the head and tail sequences of the firstoligonucleotide molecule, thereby producing a cyclic product thatcomprises the target fragment and the head and tail sequences. Thisligation step is illustrated in FIG. 1 (although, as illustrated inFIGS. 3 and 4, the method may be implemented a variety of different waysand, as such, the nucleic acid probe used in the first step of themethod can be composed of one or two oligonucleotides).

Circularlized products provide a significant advantage for detectionbecause they can be amplified by rolling circle amplification (RCA). RCAproduces hundreds or thousands of copies of a circularized product in asingle molecule, thereby effectively amplifying the circularized productand making it relatively easy to detect using, e.g., labeledoligonucleotides that hybridize to a motif in the product.

As illustrated in FIG. 1, the method may further comprise amplifying thecyclic product by rolling circle amplification using a primer thathybridizes to a sequence in the nucleic acid probe (e.g., a headsequence, a tail sequence, or a sequence therebetween). In theseembodiments, the method may further comprise quantifying the number ofrolling circle amplification products produced, thereby providing anestimate of the amount of said target fragment in the sample. In theseembodiments, the products may be amplified by rolling circleamplification using primer that is complementary to a sequence somewherein the cyclic product) to produce a plurality of RCA products, e.g.,product corresponding to a single, “cloned” fragment. The number ofrolling circle amplification products can be estimated by, e.g.,distributing the RCA products on the surface of a support (a slide),hybridizing the RCA products using labelled oligonucleotides (e.g.,fluorescently labelled oligonucleotides) and then counting the number ofdiscrete signals in an area of the support by microscopy, e.g.,fluorescence microscopy. The labelling can be done before or after theproducts have been distributed on the support and, because each RCAproduct contains thousands of copies of the same sequences, there shouldbe thousands of binding sites for the labelled oligonucleotides, therebyincreasing the signal. In multiplex embodiments (e.g., in which RCAproducts corresponding to two different chromosomes are being counted),the RCA products corresponding to one chromosome can be labelled withone fluorophore and the RCA products corresponding to another chromosomecan be labelled with a different fluorophore, thereby allowing thedifferent RCA products to be separately counted.

Quantifying signals from individual RCA products is significant because,in many applications (e.g., non-invasive pre-natal diagnosis by analysisof cell free DNA), the number of fragments corresponding to particularchromosomes (e.g., chromosome 21) needs to be determined quireaccurately and without bias. Typical analysis methods use PCR which, asis well known, is a very biased procedure in that some sequences areamplified much higher efficiencies than others. This makes PCR-basedstrategies impractical for many diagnostic efforts.

In alternative embodiments and as illustrated in FIG. 1, the targetfragment may be amplified by PCR and quantified. As would be apparent,the flanking sequences that are added to the target fragment and/or thePCR primers may be compatible with use in, e.g., Illumina's reversibleterminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLiD platform) or LifeTechnologies' Ion Torrent platform. Examples of such methods aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby etal (Methods Mol Biol. 2009; 513:19-39) and Morozova (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps. In these embodiments, the cyclic products may be amplifiedand sequenced, and the abundance of the fragments in the sample can beestimated by counting the number of sequence reads corresponding to thefragments.

In certain embodiments and as illustrated in FIG. 3, the splint sequencemay in a different molecule to the head and tail sequences, i.e., a“second” oligonucleotide molecule. As such, the the nucleic acid probeused at the beginning of the method may be composed of twooligonucleotides (a “backbone” and a “targeting” oligonucleotide, asillustrated in FIG. 3).

In other embodiments and as illustrated in FIG. 4, the splint sequencemay be in the same molecule as the head and tail sequences, i.e., in the“first” oligonucleotide molecule. As such, the nucleic acid probe usedat the beginning of the method may be composed of a singleoligonucleotide.

The target-complementary sequence may be of any length, depending on thelength of the target complementary sequence in the nucleic acid probe.In some embodiments, the target-complementary sequence is 10 to 100,e.g., 10 to 50 or 10 to 30 nucleotides in length. As noted below, thetarget-complementary sequence contains one or more mismatches (e.g., 1,2, 3, 4, 5 or 6 or more, up to 10 or more) to the target fragment and,in certain cases, the reverse complement of the target-complementarysequence may be at least 80%, at least 90% or at least 95% identical tothe target fragment.

The flanking sequences may be of any length, depending on design. Insome embodiments, the flanking sequences are 10 and 40 nucleotides,e.g., 10 and 30 nucleotides, in length.

In some embodiments, the sample may contain fragments of genomic DNA,e.g., genomic DNA from virtually any organism, including, but notlimited to, plants, animals (e.g., reptiles, mammals, insects, worms,fish, etc.), tissue samples, bacteria, fungi (e.g., yeast), phage,viruses, cadaveric tissue, archaeological/ancient samples, etc. Incertain embodiments, the genomic DNA used in the method may be derivedfrom a mammal, where in certain embodiments the mammal is a human. Inexemplary embodiments, the genomic sample may contain genomic DNA from amammalian cell, such as, a human, mouse, rat, or monkey cell. The samplemay be made from cultured cells or cells of a clinical sample, e.g., atissue biopsy, scrape or lavage or cells of a forensic sample (i.e.,cells of a sample collected at a crime scene). In particularembodiments, the nucleic acid sample may be obtained from a biologicalsample such as cells, tissues, bodily fluids, and stool. Bodily fluidsof interest include but are not limited to, blood, serum, plasma,saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid, tears,lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovial fluid,urine, amniotic fluid, and semen. In particular embodiments, a samplemay be obtained from a subject, e.g., a human. In some embodiments, thesample analyzed may be a sample of cell-free DNA obtained from blood,e.g., from the blood of a pregnant female. In certain embodiments, thegenomic DNA may be amplified, e.g., using a whole genome amplificationmethod, prior to fragmentation.

In embodiments, in which the splint sequence is in a secondoligonucleotide molecule (as shown in FIG. 3), the secondoligonucleotide may additionally comprise a capture moiety that can beemployed to enrich for the cyclic product. In these embodiments, themethod may comprise: c) immobilizing the cyclic product by binding thecapture moiety to a solid phase; and d) washing the solid phase toremove unligated nucleic acid and other reaction components; therebyenriching for the cyclic product. For example, the secondoligonucleotide may contain a biotin moiety, e.g., biotin or a biotinanalogue such as desthiobiotin, oxybiotin, 2′-iminobiotin,diaminobiotin, biotin sulfoxide, biocytin, etc., with or without alinker, e.g., -LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEGn-Biotinwhere n is 3-12, and the cyclic products can be enriched using asubstrate that is coupled to streptavidin. Biotin binds to streptavidinwith an affinity of at least 10⁻⁸M.

For non-invasive pre-natal testing embodiments, the target fragment maybe from human chromosome 21, 13 or 18.

In some embodiments, the method comprises hybridizing the sample with aset of at least 50 (e.g., at least 100, at least 200, at least 500, atleast 1,000, at least 2,000 or at least 5,000) of said probes, whereinsaid probes target different fragments on the same chromosome (e.g.,human chromosome 21, 13 or 18), and wherein the method results in aplurality of cyclic products that comprises the target fragments. Thenumber of cyclic products produced can be quantified by, e.g.,amplifying them using RCA and counting the number of RCA products, asdescribed above.

In some embodiments, the method comprises hybridizing the sample with afirst set and a second set of said sets of nucleic acid probes, whereinthe first and second sets of probes target (i.e., hybridize to fragmentsof and ligate to produce cyclic products, as described above) a firstchromosome in the sample and a second chromosome in the sample,respectively, amplifying the cyclic products by rolling circleamplification (RCA) and comparing the number of RCA productscorresponding to the first chromosome to the number of RCA productscorresponding to the first chromosome, thereby providing an estimate ofthe relative amounts of DNA from the chromosomes in the sample.

In some embodiments, the method comprises hybridizing the sample with afirst set and a second set of said sets of nucleic acid probes, whereinthe first and second sets of probes target (i.e., hybridize to fragmentsof and ligate to produce cyclic products, as described above) a firstregion and a second region of a chromosome in the sample, respectively,amplifying the cyclic products by rolling circle amplification (RCA) andcomparing the number of RCA products corresponding to the firstchromosomal region to the number of RCA products corresponding to thesecond chromosomal region, thereby providing an estimate of the relativeamounts of DNA from the chromosomal regions in the sample. Thisembodiment may be used to identify, e.g., deletions or duplications, forexample.

Also provided herein is composition comprising a nucleic acid probecomprising: i. a head sequence and a tail sequence, wherein the head andtail sequences are at opposite ends of a first oligonucleotide molecule;and ii. a splint sequence comprising, in order: an upstream flankingsequence that is complementary to the head sequence, a targetcomplementary sequence that is complementary to a target fragment in thehuman genome; and a downstream flanking sequence that is complementaryto the tail sequence; wherein the probe is designed so that, when thefirst oligonucleotide, the splint sequence, and the target fragment arehybridized to one another, the ends of the target fragment are ligatablyadjacent to the ends of the head and tail sequences in the firstoligonucleotide molecule. In certain embodiments, the composition maycomprise a first set of at least 50 (e.g., at least 100, at least 200,at least 500, at least 1,000, at least 2,000 or at least 5,000) of thenucleic acid probes, wherein the target complementary sequences of theprobes are complementary to different target fragments of a first humanchromosome (e.g., chromosome is 21, 13 or 18).

In certain embodiments, the composition may comprise a second set of atleast 50 (e.g., at least 100, at least 200, at least 500, at least1,000, at least 2,000 or at least 5,000) of said nucleic acid probes,wherein the target complementary sequences of the probes in the secondset of probes are complementary to different target fragments of asecond human chromosome. In some embodiments, the first human chromosomemay be chromosome 21 and the second human chromosome may be chromosome13 or 18. In some cases, the second human chromosome is not chromosome21, 13 or 18.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 schematically illustrates one embodiment of the subject method inwhich a circular DNA molecule is formed and amplified by RCA or PCR.

FIG. 2 schematically illustrates one embodiment of the subject method inwhich a linear ligation product is formed and enriched with solid-phasereagents.

FIG. 3 shows a probe comprising a circularised backbone oligonucleotidebound to its target fragment. The probe is illustrated in two versions,A and B.

FIG. 4 shows a circularised single oligonucleotide probe with boundtarget fragment.

FIG. 5 shows a circularised double looped probe composed of a targetingoligonucleotide and a looped backbone oligonucleotide, with bound targetfragment.

FIG. 6 shows a linear looped probe composed of a targetingoligonucleotide and a linear backbone oligonucleotide, with bound targetfragment.

FIG. 7 shows a linear probe comprising two backbone oligonucleotides,with bound target fragment.

FIG. 8 is an image of a gel showing the specificity of the methoddescribed herein.

FIG. 9 is a graph showing the precision of the method described herein.

FIG. 10 panel A shows an image of labeled RCA products on the surface ofa slide; panel B shows how ratios of fragments from differentchromosomes can be accurately determined by counting individual RCAproducts.

DETAILED DESCRIPTION The Target Nucleic Acid Fragment

The target fragment which is bound by the probe is a single strandedfragment of nucleic acid. In some embodiments, the present methods bindtarget fragments whose sequence is pre-defined. The sequence of theentire fragment including the ends may be known. Known fragments ofpre-defined sequence can be produced by specific, rather than random,fragmentation of nucleic acid. Specific fragmentation methods includedigestion with restriction enzymes, PCR (e.g., multiplex PCR), and othermethods of sequence directed fragment end definition, including otherenzymes, ribozymes, or a combination of such techniques.

One method of fragmentation is digestion with a restriction endonucleaseor a combination of two or more restriction endonucleases. Thus, thesample of fragmented nucleic acid may be a restriction enzyme digest andthe target fragment may be a restriction fragment.

A variety of specific nucleic acid cleaving enzymes are known and anysuitable enzyme may be used in the present invention, including enzymeswhich cleave at a pre-defined position within a specific nucleic acidsequence, or endonucleolytic enzymes which cleave either after or beforea specific nucleic acid recognition sequence and nicking enzymes.Catalytic nucleic acids, such as ribozymes, can be used as well for DNAfragmentation. The enzymes may cleave double stranded nucleic acid toproduce a blunt end or a sticky end, or may cleave a single strand ofnucleic acid. Various types of restriction enzymes are known, includingType I, Type II, Type III, Type IV and Type V. Suitable enzymes orcombinations of enzymes can be selected for use in the method asdesired. For example, nucleic acid in a sample (e.g. 10 ng of DNA) maybe digested with restriction enzyme (e.g. 1 U) in correspondingcompatible restriction enzyme buffer. The reaction may be incubatedunder suitable conditions (e.g. 37° C. for 1 hour), followed byenzymatic deactivation (e.g. at 80° C. for 20 minutes).

Another convenient method of providing the fragmented nucleic acid is touse primers for amplification of specific linear sequences from thenucleic acid. Multiplex PCR can be used, treating the nucleic acid withmultiple specific primer pairs to amplify multiple specific fragments.In this case, the ends of the target fragment correspond to thesequences of the pair of primers.

For many diagnostic and other applications, the sample is a sample offragmented chromosomes (e.g., human chromosomes or microbialchromosomes). The target fragment may be a genome fragment specific toone chromosome of an organism's genome. In other words, the targetfragment may be found only in one chromosome of the genome and not inother chromosomes of that genome. Commonly, the method will be used foranalysis of the human genome, in which case the target fragment may be afragment specific to one human chromosome, i.e., found in thatchromosome and not in other human chromosomes. For example, the fragmentmay be specific to chromosome 21.

The target fragment may be specific to one locus of a chromosome.Accordingly, it may be found in that chromosomal locus and not in otherloci of the same chromosome or other chromosomes of the same genome. Forexample, the fragment may be specific to one locus of a humanchromosome.

A given species of nucleic acid in a sample may encompass somevariability, for example a sample may comprise chromosomes of differentindividuals, such as nucleic acid obtained from maternal blood whichcontains maternal DNA and foetal DNA. Here the species of interest maybe a particular chromosome, but it is convenient to detect all copies ofthat chromosome whether of foetal or maternal origin. Thus, a species ofinterest may be one chromosome or chromosomal locus, and the targetsequences are found in that chromosome or locus in both maternal andfoetal copies of the chromosome or chromosomal locus.

Samples of nucleic acid may be provided in any suitable way, for exampleas samples of biological tissue or fluid from patients. Samples may beblood samples, whole blood, plasma, or serum, tissue samples, e.g.,formalin fixed paraffin embedded samples of tissue, or may be samples ofnucleic acid extracted from blood or tissue.

The sample may be any sample that contains nucleic acid. The nucleicacid contained in the sample may be DNA and/or RNA. The sample may becomplex, e.g. whole genomic DNA, or cDNA from a whole organism, tissueor cell population, or a fraction thereof. In this regard it may, forexample, be a direct product of a nucleic acid isolation procedure, orof a cell lysis procedure, or it may be further be fractionated orpurified in some way, e.g. it may contain nucleic acids which have beenpartially or fully separated in some way, or treated in any way, e.g.RNA to produce cDNA. The sample may be from any eukaryotic orprokaryotic or viral source, e.g. may be microbial (for examplebacterial or fungal), plant, or animal. Preferably the sample is ofhuman origin, e.g., human genomic DNA. The sample may be a tissue orblood sample from an animal, where the nucleic acid to be detected ismicrobial, e.g., bacterial, viral or fungal. For methods relating tonon-invasive prenatal diagnostics, the sample is derived from the bloodof a pregnant woman and comprises foetal DNA. In other examples, thenucleic acid to be detected or quantified is tumour associated DNA.

Usually, the method may be performed on the samples in vitro.Accordingly, the methods generally do not include diagnosis carried outin vivo on the human or animal body or methods of treatment of the humanor animal body by surgery or therapy. Nevertheless, the results of thein vitro diagnostic methods may be used to inform the subsequenttreatment of patients.

Denaturing the Target Nucleic Acid

The probe recognises and binds the target nucleic acid in singlestranded form, through hybridisation between the single strandedfragment and the target-complementary sequence of the targetingoligonucleotide. Therefore, if the target fragment in the sample is notalready single stranded, denaturing conditions should be provided toseparate the single stranded target fragment from its complementarynucleic acid strand.

The denaturing conditions may be a sufficiently high temperature toseparate the target fragment from its complementary sequence. Denaturingconditions may be incubation at 95° C. for a suitable time, e.g. 10minutes. Alternatively chemical denaturation may be performed.

Complementarity

A method of testing a sample for the presence of a target fragment maycomprise contacting the sample with a nucleic acid probe, wherein theprobe comprises

a targeting oligonucleotide containing a target-complementary sequence,which is the complement of the target fragment, and a flanking sequenceadjacent to the target-complementary sequence and

an oligonucleotide sequence having a free 5′ or 3′ end, wherein theoligonucleotide sequence is complementary to the flanking sequence.

Suitable concentrations of probes may be determined based on theconcentration (or expected concentration) of the target fragment ortarget fragments in the sample. As illustrated in the Examples, probesmay be added to the sample at a concentration of 10 pM per probe. Wherea sample is contacted with multiple probes (e.g. a set of probes),concentrations of the individual probes may be 10 pM. Preferably, probesare used in excess of the expected concentration of the nucleic acidspecies of interest to be detected or quantified. Use of excess probeshould ensure that all copies of target sequences present in the sampleare recognised. This maximises the sensitivity of detection. Also, wheremethods involve quantification, it ensures that the detection of theligation products or cumulative signal from a set of probes isproportional to the quantity of target sequences in the sample.

Under annealing conditions, the target fragment (if present) hybridisesto the target complementary sequence of the targeting strand and theoligonucleotide sequence hybridises to the flanking sequence, so thatthe free 5′ or 3′ end of the oligonucleotide sequence is injuxtaposition with the 3′ or 5′ end of the target fragment respectively.Thus, the targeting oligonucleotide templates the target fragment forligation to the oligonucleotide sequence. The 3′ end of the targetfragment may be ligated to a 5′ end of a head sequence and the 5′ end ofthe target fragment may be ligated to a 3′ end of a tail sequence in adouble ligation event.

In probes according to the present invention, maximum specificity forthe target fragment is achieved if the target-complementary sequence isthe exact complement of the target fragment, so that there is perfecthybridisation between them. However, this is not essential in all cases,and a small degree of mismatching may be accepted, for example to allowdetection of fragments which exhibit allelic variation where it isdesired to detect the fragment regardless of the exact allele present inthe sample. Alternatively, multiple probes can be designed for variantsequences. This can enable both detection and discrimination ofdifferent alleles or mutations. Probes according to the presentinvention are most advantageously used in multiplex methods where largenumbers of different probes are included in a reaction. Within such aplurality of probes, it is envisaged that the majority of probes willhave perfect complementarity for their target fragments but some probesmay bind targets with minor mismatches.

Preferably, the target-complementary sequence has fewer than 5 base pairmismatches with the target fragment. There may optionally be one, two,three or four base pair mismatches between the target fragment and thetarget-complementary sequence. A mismatch may be a point at which acorresponding base is absent from one sequence, so that thecomplementary sequence forms a loop at the mismatched point, or mayoccur where a non-complementary nucleotide is present in one sequenceand so does not pair with the base at the corresponding position of theother sequence. Where there is an incorrect base pairing, i.e., apairing of A or T with C or G, hydrogen bonding does not take placebetween the bases of the two strands, although hybridisation will stilltake place between the target fragment and the target complementarysequence of the targeting oligonucleotide due to base-pairing betweenthe nucleotides neighbouring the mismatch. Mismatches may be wobblebases. A wobble base would normally correspond to a position in thetarget complementary sequence that pairs with a position of knowngenetic variation in the target fragment. The probe may be synthesisedby adding one or several dideoxynucleotides during the specificsynthesis cycle for the wobble base position. This is typically the casefor traditional oligonucleotide synthesis. Alternatively multipleseparate probes may be produced, one for each genetic variant. This istypically the case if probes are synthesised using microarray basedsynthesis. A wobble base may correspond to single nucleotide differencesbetween codons, where the different codons encode the same amino acid.

In general, longer target-complementary sequences for hybridising longertarget fragments may tolerate a higher number of mismatches comparedwith shorter target-complementary sequences. The target-complementarysequence may, for example, have at most 1 in 8, 1 in 9 or 1 in 10 basepair mismatches with the target fragment. Any such mismatches should berestricted to the internal region of the target complementary sequenceand target fragment, so that they do not inhibit ligation or sequencespecific target fragmentation by e.g. restriction enzyme digestion.Accordingly, preferably there is perfect complementarity between thetarget fragment and the target complementary sequence in the terminal 6to 8 nucleotides, preferably the terminal 10 nucleotides at each end ofthe target fragment.

Generally, the target fragment and the target-complementary sequence areof the same length. The full length of the target fragment is thus boundby the target complementary sequence. Hybridisation of the targetfragment to the targeting oligonucleotide represents a single bindingevent between the two nucleic acid molecules, contrasting with probeswhich bind the two ends of a target molecule or to two non-adjacentregions of the target.

The target-complementary sequence may have a length of at least 10nucleotides, for example at least 15 nucleotides. It may be up to 20,25, 30, 35 or 40 nucleotides long. Preferred ranges include 10-20nucleotides, 10-30 nucleotides, and 10-40 nucleotides. Such relativelyshort target-complementary sequences are suitable for bindingcorrespondingly short fragments. The short sequence contributes to thespecificity of the double ligation reaction, since DNA ligase issensitive to base pair mismatches and will preferentially ligateperfectly matched sequences. Where mismatches are present in thefootprint of DNA ligase bound to the double stranded sequence, thesequences may not be ligated, which provides an additional proofreadingstep ensuring high specificity in detecting the target fragment inpreference to fragments of different but similar sequence. DNA ligasetypically has a footprint of 6 to 8 bases on each side of the nick.Therefore, if the fragment is 20 bases, 12 to 16 of the bases will becovered by ligase specificity.

The probe hybridisation will discriminate against mismatches especiallyin the central part of the hybridised sequence while the ligation willdiscriminate against mismatches at the ends of the target fragment.Together this generates a highly specific fragment detection.

The targeting oligonucleotide is longer than the target fragment sinceit includes the flanking sequences as well as the target-complementarysequence, and it may further include one or more custom sequences. Acustom sequence is not complementary to other regions of the probe or tothe target fragment—in other words it does not hybridise to otherregions of the probe (outside the custom sequence) or to the targetfragment under annealing conditions. The upstream flanking region isupstream of or 5′ of the target-complementary sequence in the targetingoligonucleotide. The downstream flanking region is downstream of or 3′of the target-complementary sequence in the targeting oligonucleotide.Accordingly, the target-complementary sequence is internal to thetargeting oligonucleotide and does not include an end of the targetingoligonucleotide, since it is flanked by the upstream and downstreamflanking sequences.

The double stranded sequence produced by hybridisation of the targetfragment and the target-complementary sequence may be considered ahybrid double stranded sequence, since it is a hybrid of the target andthe probe. Typically the double stranded sequence adopts a doublehelical conformation, in which the target fragment is one strand and thetargeting oligonucleotide is the other strand of the double helix. Thehybrid double stranded sequence is flanked by the upstream anddownstream flanking sequences of the targeting oligonucleotide, which inturn hybridise to the head and tail sequences to produce double strandedsequences. Again, these typically adopt the normal double helicalconformation of double stranded nucleic acid.

The upstream and downstream flanking sequences are preferably differentfrom each other, i.e., preferably have different sequences. It ispreferred that the head sequence is complementary to the upstreamflanking sequence but not to the downstream flanking sequence, and thatthe tail sequence is complementary to the downstream flanking sequencebut not to the upstream flanking sequence. This ensures that the headand tail sequences hybridise only to the upstream and downstreamflanking sequences respectively.

The head sequence will usually be the same length as the upstreamflanking sequence. The tail sequence will usually be the same length asthe downstream flanking sequence.

Normal lengths for the flanking sequences are between 10 and 40nucleotides, for example 10-20 or 10-30 nucleotides. The flankingsequences may be the same length as each other. One or both flankingsequences may be the same length as the target-complementary sequence.The upstream and/or downstream flanking sequence may thus have a lengthof at least 10 nucleotides, for example at least 15 nucleotides. It maybe up to 20, 25, 30, 35 or 40 nucleotides long.

Preferably, the head sequence is the complement of the upstreamsequence. Preferably, the tail sequence is the complement of thedownstream sequence. Perfect matching of the sequences is desirable foroptimum binding of the probe so that the head and tail sequences arecorrectly positioned for ligation to the target fragment. Optionally,however, there may be one, two three or four base pair mismatchesbetween the head sequence and the upstream flanking sequence, and/orbetween the tail sequence and the downstream flanking sequence.Preferably, there are fewer than 5 base pair mismatches.

Other than the target-complementary sequence, the probe should usuallynot be complementary to the target fragment or to other nucleic acidsthat may be present in the sample. This is to avoid unwantedhybridisation of the probe to nucleic acid other than the target. Thus,if the probe is for binding a fragment of human genomic DNA, the probemay be designed so that sequences other than the target-complementarysequence are not complementary to human genomic DNA, so that the probeonly hybridises to the target fragment and not to other nucleic acid inthe sample.

Annealing and Ligation

The target fragment is ligated in a highly specific reaction at bothends. Since the target fragment is typically the product of a specificfragmentation of nucleic acid, these ends will usually have a specific,pre-determined sequence. In the ligation step, these ends arespecifically detected by sequence-dependent ligation to the head andtail sequences respectively. Preferably, binding of the target fragmentto the probe creates two perfectly matched ligatable junctions, onebetween the 3′ end of the target fragment and the 5′ end of the headsequence and one between the 5′ end of the target fragment and the 3′end of the tail sequence.

Ligation of a 5′ end of nucleic acid to a 3′ end of nucleic acid canoccur when the two ends are base paired to adjacent nucleotides of acomplementary sequence. Base pairing of the respective end nucleotidesto the adjacent nucleotides forms a nucleic acid strand containing anick between the two ends. Ligation of the two ends can be catalysed byDNA ligase. Providing conditions for ligation will therefore usuallycomprise providing a DNA ligase enzyme and reaction conditions underwhich the DNA ligase ligates the two ends to form a continuous nucleicacid strand, closing the nick. A number of ligase enzymes arecommercially available, such as Ampligase (Epicentre), for whichsuitable conditions are to add 1 U enzyme and incubate at 55° C. for 1hour in ligase buffer.

The targeting oligonucleotide templates the target fragment for ligationto the head and tail sequences, due to the location of thetarget-complementary sequence between the flanking sequences. Underannealing conditions in the presence of the target fragment, the headand tail sequences hybridise to the flanking sequences, defining a gapbetween the 5′ end of the head sequence and the 3′ end of the tailsequence. The target fragment hybridises to the target-complementarysequence in the gap. Thus, hybridisation of the head and tail sequencesand the target fragment to the targeting oligonucleotide positions the3′ end of the target fragment in juxtaposition with the 5′ end of thehead sequence, and positions the 5′ end of the target fragment injuxtaposition with the 3′ end of the tail sequence.

Positioning of two ends in juxtaposition provides a substrate for DNAligase to ligate the ends together. It is preferable that the 5′ end ofthe head sequence and the 3′ end of the target fragment hybridise toadjacent nucleotides of the targeting oligonucleotide, and the 3′ end ofthe tail sequence and the 5′ end of the target fragment hybridise toadjacent nucleotides of the targeting oligonucleotide. Accordingly, theupstream flanking sequence may be immediately adjacent to thetarget-complementary sequence, with no intervening nucleotides.Similarly, the downstream flanking sequence may be immediately adjacentto the target-complementary sequence, with no intervening nucleotides.Adjacent 3′ and 5′ ends can be directly ligated by DNA ligase sealingthe nick between them to form a continuous nucleic acid strand.

The product of the double ligation, i.e., the product of ligating boththe head sequence and the tail sequence to the target fragment, is acontinuous strand of nucleic acid. It is continuous in the sense that itcontains no nicks or gaps, so all nucleotides in the strand arecovalently joined.

The probe may be designed so that the continuous strand of nucleic acidcomprising the head and tail sequences and the target fragment is acircle of nucleic acid. The term circle here refers to the topology ofthe strand being a closed loop, with no free end.

Under annealing conditions in the presence of the target fragment, thehead and tail sequences hybridise to the flanking sequences, defining agap between the 5′ end of the head sequence and the 3′ end of the tailsequence. The target fragment hybridises to the target-complementarysequence in the gap, thereby positioning the ends of the target fragmentin juxtaposition with the 5′ end of the head sequence and the 3′ end ofthe tail sequences, and completing a circle of nucleic acid whichcomprises the target fragment and the head and tail sequences.

The nucleic acid molecules which form the circle have their ends injuxtaposition. Ligation of the ends produces the continuous circularstrand of nucleic acid comprising at least the head and tail sequencesand the target fragment.

Probes which form a circle of nucleic acid include probes in which thehead and tail sequences are provided on a single nucleic acid molecule.For example, in addition to the targeting oligonucleotide the probe maycomprise a backbone oligonucleotide having the head and tail sequencesat its 5′ end 3′ ends respectively, wherein the head and tail sequencesof the backbone oligonucleotide bind in trans to the flanking sequencesof the targeting oligonucleotide under the annealing conditions. Thebackbone oligonucleotide may comprise a custom sequence between the headand tail sequences. FIG. 3 illustrates embodiments of such probes.Alternatively, the head and tail sequences of the backboneoligonucleotide may be adjacent, with no custom sequence between them.

In another example, the head and tail sequences may be at ends of thetargeting oligonucleotide and bind in cis to the flanking sequencesunder the annealing conditions. The targeting oligonucleotide maycomprise a custom sequence between the targeting oligonucleotide and thehead and/or tail sequence. FIG. 4 illustrates an embodiment of such aprobe.

Probes which form a circle of nucleic acid also include probes in whichthe head and tail sequences are provided on different nucleic acidmolecules. In such cases, the circle of nucleic acid which forms underthe annealing conditions will comprise at least three nucleic acidmolecules—the target fragment, the head sequence and the tail sequence.The ends of the nucleic acid molecules will all be in juxtaposition, aspreviously noted. More than two ligation reactions are required to formthe continuous circular strand of nucleic acid in such cases. An exampleis where the tail sequence is the 3′ end of the targetingoligonucleotide, and the probe comprises a backbone oligonucleotidehaving the head sequence at its 5′ end. Under the annealing conditionsthe tail sequence binds in cis to the downstream flanking sequence ofthe targeting oligonucleotide, and the head sequence of the backboneoligonucleotide binds in trans to the upstream flanking sequence of thetargeting oligonucleotide. Binding in cis means that the binding takesplace on the same nucleic acid molecule, i.e., a single strand ofnucleic acid forms a three dimensional structure in which differentregions are brought together and hybridise. Binding in trans means thatthe binding takes place between different nucleic acid molecules.Optionally, the backbone oligonucleotide comprises a pair of invertedrepeat sequences which form a hairpin structure under annealingconditions, thereby positioning the 3′ end of the backboneoligonucleotide in juxtaposition with the 5′ end of the targetingoligonucleotide. There is a nick between the two ends. A probe of thistype is illustrated in FIG. 5. When conditions for ligation areprovided, the 5′ end of the targeting oligonucleotide is ligated to the3′ end of the backbone oligonucleotide. The product of double ligationis a circle of nucleic acid comprising the targeting oligonucleotide,the target fragment and the backbone oligonucleotide. Alternatively,where there is a gap between the 5′ end of the targeting oligonucleotideand the 3′ end of the backbone oligonucleotide, the probe shown in FIG.5 will not be circularised by ligation—instead the continuous strand ofnucleic acid comprising the head and tail sequences and the targetfragment is a linear strand of nucleic acid.

The probe may alternatively be arranged in the opposite orientation sothat the head sequence is at the 5′ end of the targeting oligonucleotideand the probe comprises a backbone oligonucleotide having the tailsequence at its 3′ end. In this case, under the annealing conditions thehead sequence binds in cis to the upstream flanking sequence of thetargeting oligonucleotide, and the tail sequence of the backboneoligonucleotide binds in trans to the downstream flanking sequence ofthe targeting oligonucleotide. Again, the backbone oligonucleotide maycomprise a pair of inverted repeat sequences which form a hairpinstructure under annealing conditions to position the 5′ end of thebackbone oligonucleotide in juxtaposition with the 3′ end of thetargeting oligonucleotide. The 3′ end of the targeting oligonucleotideis then ligated to the 5′ end of the backbone oligonucleotide so thatthe product of double ligation is a circle of nucleic acid comprisingthe targeting oligonucleotide, the target fragment and the backboneoligonucleotide. Alternatively, as noted above, the annealing mayposition the 5′ end of the backbone oligonucleotide near the 3′ end ofthe targeting oligonucleotide but separated by a gap of one or morenucleotides. The ligated product will then be a continuous linear strandof nucleic acid comprising the head and tail sequences and the targetfragment.

The backbone oligonucleotide may comprise a custom sequence between theinverted repeat sequence, so that under the annealing conditions thebackbone oligonucleotide forms a hairpin loop, as illustrated in FIG. 5.

As noted, probes may be designed so that the continuous strand ofnucleic acid comprising the head and tail sequences and the targetfragment is a linear strand of nucleic acid. Under annealing conditionsin the presence of the target fragment, the head and tail sequenceshybridise to the flanking sequences, defining a gap between the 5′ endof the head sequence and the 3′ end of the tail sequence. The targetfragment hybridises to the target-complementary sequence in the gap,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tailsequences, and completing a strand of nucleic acid which comprises thetarget fragment and the head and tail sequences. The nucleic acidmolecules which form the strand have their ends in juxtaposition. Theterm juxtaposition has been discussed elsewhere. There is a nick betweenthe ends to be ligated. Ligation of the ends produces the continuousstrand of nucleic acid comprising at least the head and tail sequencesand the target fragment.

The probe may comprise a targeting oligonucleotide having the tailsequence at its 3′ end and a linear backbone oligonucleotide having thehead sequence at its 5′ end. Under annealing conditions, the tailsequence binds in cis to the downstream flanking sequence of thetargeting oligonucleotide, and the head sequence of the backboneoligonucleotide binds in trans to the upstream flanking sequence of thetargeting oligonucleotide. The targeting oligonucleotide may comprise acustom sequence between the downstream flanking sequence and the tailsequence, so that under the annealing conditions the targetingoligonucleotide forms a hairpin loop. The linear strand of nucleic acidformed under annealing conditions comprises the backboneoligonucleotide, the target fragment and the targeting oligonucleotide.FIG. 6 illustrates this arrangement.

The probe may equally be arranged in the reverse orientation, where thehead sequence is at the 5′ end of the targeting oligonucleotide, and theprobe comprises a backbone oligonucleotide having the tail sequence atits 3′ end. In this case the head sequence binds in cis to the upstreamflanking sequence of the targeting oligonucleotide and the tail sequenceof the backbone oligonucleotide binds in trans to the downstreamflanking sequence of the targeting oligonucleotide.

Another form of probe which forms a linear nucleic acid strand as theproduct of ligation is a probe comprising the head and tail sequences onseparate backbone oligonucleotides. Such a probe may comprise a backboneoligonucleotide comprising a head sequence having a free 5′ end, and abackbone oligonucleotide comprising a tail sequence having a free 3′end, wherein under the annealing conditions the head and tail sequencesbind in trans to the flanking sequences of the targetingoligonucleotide. One or both backbone oligonucleotides may furthercomprise a custom sequence. FIG. 7 illustrates probes of this type.

Preferably, the oligonucleotides of the probe in its unligated form arelinear. So, preferably the targeting oligonucleotide is a linear nucleicacid molecule. For probes including one or more backboneoligonucleotides, these are also preferably linear. This allowsconvenient differentiation between ligated and unligated probes where acircle of DNA is formed only as a result of successful ligation of thecircularising embodiments of the probe. Linear nucleic acid moleculesare not amplified by rolling circle replication.

Detection

After providing conditions under which the target fragment, if present,is ligated to the probe, a detection step is performed to determinewhether or not such ligation has occurred. This indicates whether or notthe target fragment was present in the sample. Thus, detection ofproduct is dependent on successful ligation of the target fragment tothe head and tail sequences to form the continuous strand of nucleicacid. The detection step therefore generally involves detecting a signalthat requires the presence of both ligation junctions. For example,detection may comprise amplification across both ligation junctions(e.g., by PCR or, for circularising embodiments of the probe, rollingcircle replication), or capturing the continuous nucleic acid strand atone end and detecting its other end.

Optionally, a method may include enriching the product of doubleligation before detection. Products may be enriched by amplificationand/or by solid phase chemistry. Circular nucleic acid products may beselectively enriched by treating the sample with exonuclease (e.g.,Lambda exonuclease) to digest linear nucleic acid products. In general,exonuclease degradation may be used to enrich for ligation products whenthe ligation products are protected from exonuclease degradation.Exonuclease should then be deactivated (e.g. by heat) before anysubsequent step involving polymerisation, e.g. before rolling circleamplification. As illustrated in Example 1, 1 U Exonuclease may be addedto remove non-reacted probes and fragments. Suitable conditions areincubation at 37° C. for 1 hour in corresponding exonuclease buffer,followed by enzyme inactivation at 80° C. for 20 minutes. Wherecapture/detect methods are used, ligation products may be enriched bycapturing the products on a solid phase via the capture moiety. Asillustrated in Example 2, a solution containing linear ligation productsmay be mixed with 10 ml M-280 streptavidin coated magnetic beads(Invitrogen) in Tris-HCl (pH 7.5), 3.5 mM EDTA and 0.07% Tween-20 in afinal volume of 200 ml, and incubated at room temperature for 15minutes. After incubation, the beads are collected using a ring magnetand supernatant is removed. Other ways of enriching for ligationproducts include specifically size-selecting ligation products.

A convenient way to detect the product of double ligation to provideconditions for amplification and to test for the presence of theamplification product. Several amplification approaches are possible,such as NASPA, LAMP, T7 amplification, PCR or, where the continuousstrand is a circle, rolling circle replication. The step of detectingthe product of double ligation may comprise providing conditions foramplification across the first and second ligation junctions of thecontinuous strand of nucleic acid, and detecting whether anamplification product is present. Ligation products may be amplified byclonal amplification. Suitable amplification techniques include rollingcircle amplification (see below), bridge PCR (Adessi C, et al., NucleicAcids Res. 2000 Oct. 15; 28(20):E87), emulsion PCR (digital PCR inemulsions was described by Dressman et al., Proc Natl Acad Sci USA. 2003Jul. 22; 100(15):8817-22. Epub 2003 Jul. 11) and digital PCR (Vogelstein& Kinzler, Proc Natl Acad Sci USA. 1999 Aug. 3; 96(16):9236-41). Clonallocalised amplification in gels was described by Mitra & Church, NucleicAcids Res. 1999 Dec. 15; 27(24): e34.

Where the product of double ligation is a circle of nucleic acid, aconvenient way to detect the product is to provide conditions forrolling circle replication and to detect whether a product of rollingcircle replication is present. The product of rolling circle replicationis dependent on double ligation to provide the circle of nucleic acidfor amplification. Rolling circle replication was described in U.S. Pat.No. 5,854,033 (Lizardi) and Fire & Xu, Proc Natl Acad Sci USA. 1995 May9; 92(10):4641-5. Rolling circle replication is an amplification of acircular nucleic acid molecule using a strand displacing DNA polymerase,resulting in large DNA molecules containing tandem repeats of theamplified sequence. The DNA polymerase catalyses primer extension andstrand displacement in a processive rolling circle polymerisationreaction that proceeds as long as desired. It results in anamplification of the circularised probe sequence orders of magnitudehigher than a single cycle of PCR replication and other amplificationtechniques in which each cycle is limited to a doubling of the number ofcopies of a target sequence. Additional amplification can be obtainedusing a cascade of strand displacement reactions. Rolling circlereplication may be hyper branched rolling circle replication.Hyperbranched RCA was described by Lizardi et al., Nat Genet. 1998 July;19(3):225-32. Conditions for rolling circle replication are illustratedin the Examples, for example incubation with 1 U of phi29 polymerase(New England Biolabs) can be added in corresponding phi29 buffer andnucleotides (dNTPs) at 37° C. for 1 hour.

Following rolling circle replication, the amplified probe sequences canbe detected and quantified using any of the conventional detectionsystems for nucleic acids such as detection of fluorescent labels,enzyme-linked detection systems, antibody-mediated label detection, anddetection of radioactive labels. Preferably, a rolling circleamplification product is detected by hybridisation of a labelleddetection oligonucleotide to a motif in the RCA product, e.g. a motif ina custom sequence of the probe. Because the amplified product isdirectly proportional to the amount of target sequence present in asample, quantitative measurements reliably represent the amount of atarget sequence in a sample. Major advantages of this method are thatthe ligation step can be manipulated to obtain allelic discrimination,the DNA replication step is isothermal, and signals are strictlyquantitative because the amplification reaction is linear and iscatalysed by a highly processive enzyme. In multiplex assays, the primeroligonucleotide used for the DNA polymerase reaction can be the same forall probes.

For probes in which the head and tail sequences are on separate nucleicacid molecules, it may be convenient to use capture/detect methods. Theproduct of double ligation contains the head and tail sequences in asingle nucleic acid molecule (the continuous strand), whereas unligatedprobes do not. Accordingly, the product of double ligation may bespecifically detected by capturing the nucleic acid molecule containingthe head sequence, washing to remove unligated probe nucleic acid, thendetecting the presence of the tail sequence in the captured fraction.Alternatively, the product of double ligation may be detected bycapturing the nucleic acid molecule containing the tail sequence,washing to remove unligated probe nucleic acid, then detecting thepresence of the head sequence in the captured fraction. Detection isspecific to the ligated probes, since the head and tail sequences in theunligated probes are connected only by hybridisation between the nucleicacids and are separated by washing, whereas the ligated probes containthe head and tail sequences in a continuous nucleic acid strand, i.e.,covalently joined.

A probe may be modified to carry a capture moiety. The capture moietymay permit attachment to a solid substrate such as a bead. A suitablecapture moiety is biotin, which pairs with streptavidin, allowing themodified probe nucleic acid to be isolated on the solid substrate coatedwith streptavidin. Where a probe comprises a backbone oligonucleotidecontaining either the head or tail sequence, and a separate nucleic acid(targeting oligonucleotide, or a second backbone oligonucleotide)containing the tail or head respectively, either of these nucleic acidmolecules may carry a capture moiety, for example may be biotinylated.It may be convenient to provide the probe with the capture moiety beforecombining the probe with the sample. Alternatively, the capture moietymay be introduced after the ligation step.

Where one nucleic acid molecule in the probe carries a capture moiety,the other may carry a label. It is possible to use the nucleic acidsequence itself as a label, for example to specifically detect thepresence of the head sequence (where the tail is captured) or the tailsequence (where the head is captured) or to detect a custom sequencewhich is unique to the nucleic acid molecule to be detected. Acomplementary oligonucleotide may be used for detection. Alternativelythe nucleic acid may carry a heterogeneous label such as a fluorophore.The heterogeneous label is not part of the nucleic acid itself. Otherlabels that can be used include quantum dots, bioluminescence, signalgenerating enzyme cascades like tyramide signal amplification, andradioactive moieties. The method may then comprise detecting thepresence of the label, e.g., detecting fluorescence, detecting thequantum dots, detecting bioluminescence, detecting the signal generatedby the enzyme, or detecting radioactivity, respectively.

As an example, the step of detecting whether the product of doubleligation is present may comprise capturing a backbone oligonucleotide ofthe probe on a substrate via the capture moiety, washing the substrateto remove unligated probes and retaining a captured fraction comprisingthe substrate and captured backbone oligonucleotide, and testing for thepresence of the product of double ligation in the captured fraction.Where the product of double ligation carries a label, this may comprisetesting for the presence of the label in the captured fraction. Thecapture moiety can be a biotin-molecule with affinity to a streptavidinsubstrate. Other suitable affinity tags include polyhistidine tags withaffinity to immobilised metal ions, such as cobalt, nickel, copper whichcan be used for the purification of histidine containing sequences,e.g., backbone oligonucleotides. The capture moiety may thus be part ofthe sequence to be captured, e.g. a His-tag sequence, or it may be aheterogenous moiety which is not part of the nucleic acid itself.

A suitable solid substrate is a bead, for example magnetic beads tofacilitate enrichment of the captured products using a magnet. Thesubstrate may be coated with a binding member for the capture moiety,e.g. streptavidin coated magnetic beads may be used with biotinylatedprobes.

An advantage of some embodiments of the present method is that it do notrely on nucleic acid sequencing, nor PCR, which causes biased resultsbecause some sequences amplify more efficiently than others. Optionallythough, detection may comprise a step of validating the identity of theligated fragment by sequencing the product. One of the advantages of thepresent invention is that, by incorporating the actual target fragmentin the product of double ligation, the product can be sequenced toconfirm that the probes reacted with the correct target. This is anadvantage compared with other approaches based on double ligation suchas US20130172212 (Ariosa).

Multiplexing

Multiple different target nucleic acid fragments may be detected using aplurality of the probes in parallel. For example, a sample of fragmentedchromosomes may be contacted with a set of probes for binding multiplefragments of a chromosome, wherein each probe in the set is for bindinga different target fragment specific to that chromosome. The probes mayshare a common custom sequence, which can be used as a barcode toidentify the probes that specifically bind that chromosome. Multiplexingcan include multiplex targeting oligonucleotides and one common backboneoligonucleotide but also several sets of targeting oligonucleotideswhere each subset hybridises to separate backbone oligonucleotides.

Multiple probes can be used to provide a detectable signal, where themagnitude of the signal is proportional to the number of probesrecognising their target fragments. Individual signals from theplurality of probes are converted into a single cumulative detectablesignal, amplifying the individual signals through the multiplex probing.Ten or more probes produce a signal amplification of ten-fold or more.The generated signals depend on correctly reacted probes upon targetrecognition, using sequence specific hybridisation and ligation togenerate the specific products of double ligation from which the signalis obtained.

Each probe that recognises its target fragment generates a ligationproduct, and the ligation products produced by each probe hybridisationmay be individually detectable, so that an individual signal isobtainable from each. However, an elegant feature of the presentinvention is that these individual signals need not be individuallydetected, but instead are merged into a cumulative signal and thecumulative signal is detected. The cumulative signal is a combination ofthe individual signals and can thus be used to detect and/or quantifythe ligation products, representing the presence or quantity of thenucleic acid species under investigation. Some implementations of thepresent method allow an earlier merging of the probe signals comparedwith methods involving sequencing and microarrays, in which individualsignals are generated for multiple probes across a region and then thesignal is merged in the analysis to represent a region. The signal canbe merged before detection, so that individual signals are notseparately mapped or interrogated. This enables a simpler readoutformat.

An individual signal may be obtainable from each product of doubleligation which is formed as a result of probe hybridisation to eachtarget fragment. So, for example, where a set of probes comprises 10different probes that recognise 10 target fragments of a species ofinterest in a sample, there will be 10 ligation products includingligation junctions, and a cumulative signal may be detected, which isthe combination of individual signals from the 10 ligation products. Ofcourse, in this example the actual number of molecules probes, targetfragments and ligation products may be higher than 10 because there willusually be multiple copies of each target fragment in a sample and thesample will be contacted with multiple copies of each probe.

Method of signal amplification by multiplexing can be used to detectnucleic acid species of interest in a sample, for example where anucleic acid species is a minor or trace component in a complex nucleicacid sample. The amplification by multiplexing enables reliabledetection. This may be used for example to detect microbial nucleic acidin samples, such as patient samples, for diagnostic purposes. Samplesmay be probed with probes specific for microbial nucleic acids ofmultiple species, to detect and identify those present. This is usefulfor detection of agents of infectious disease, such as bacteria, virusesand fungi. Specific nucleic acid transcripts may be detected.Amplification by multiplexing may also be used to quantify the nucleicacid species. By probing two or more species of nucleic acid—one or morespecies of interest and one or more reference nucleic acid species—themethod enables quantification of the relative amounts of the two speciesin the sample. The method is especially useful when applied to thedetection or quantification of chromosomes or chromosomal loci, forexample for chromosomal copy number detection. An application ofparticular value is the use of such methods for identifying chromosomaldefects, including for the diagnosis of cancers and congenitalaneuploidies. Use for non-invasive prenatal diagnosis (NIPT) isspecifically described.

A species of nucleic acid in a sample may be detected by contacting thesample with a set of probes according to the present invention, whereineach probe specifically recognises a distinct target sequence in thespecies of nucleic acid to be detected. The target sequences correspondto target fragments of the species of nucleic acid. Recognition of eachtarget sequence by each probe generates a product of double ligation asdescribed herein. A cumulative signal can then be detected, this being acombination of signals from the products. Detection of the signalindicates the presence of the species of nucleic acid in the sample. Thespecies of nucleic acid may be quantified by quantifying the cumulativesignal to determine a signal level, wherein the signal level isproportional to the quantity of the species of nucleic acid in thesample, and thereby determining the quantity of the species of nucleicacid in the sample. A first species of nucleic acid may be quantifiedrelative to a second or reference species of nucleic acid by contactingthe sample with a first set of probes and a second set of probes,wherein the probes of the first set each specifically recognise adistinct target sequence within the first species of nucleic acid andwherein the probes of the second set each specifically recognise adistinct target sequence within the second or reference species ofnucleic acid. First and second cumulative signals are detected, thefirst cumulative signal being a combination of individual signals fromproducts generated by probes of the first set recognising their targetsequences, and the second cumulative signal being a combination ofindividual signals from products generated by probes of the second setrecognising their target sequences. The first and second signals arequantified to determine first and second signal levels respectively,these being proportional to the quantities of the first and secondspecies of nucleic acid in the sample. The relative quantities of thefirst and second nucleic acid species in the sample may thus bedetermined by comparing the first and second signal levels.

For example, the cumulative signal may be the summarised enumeration ofclonally amplified and/or labelled products of the probes that recognisetheir target sequences, for example products of rolling circleamplification, or a fluorescent signal emitted from all the productswhere each product emits a fluorescent signal. For quantifying relativeamounts of multiple species of nucleic acids, different signals are usedfor each species, for example products of one set of probes may emit adifferent wavelength or spectrum of fluorescence compared with productsof another set of probes.

A species of nucleic acid in a sample may be detected in a method,comprising contacting the sample with a set of probes, wherein eachprobe specifically recognises a distinct target sequence within thespecies of nucleic acid to be detected, providing denaturing conditionsunder which the target sequences in the species of nucleic acid aresingle stranded, providing conditions for annealing and ligation, underwhich conditions the probes hybridise to their target sequences andgenerate ligation products, and detecting a cumulative signal which is acombination of individual signals from all ligation products, whereindetection of the signal indicates the presence of the species of nucleicacid in the sample.

Details of the sample, target nucleic acid, method steps (e.g.,denaturing, annealing, ligation) and probes are described elsewhereherein. The method may comprise:

(i) providing a sample in which the species of nucleic acid isfragmented into target fragments,(ii) providing denaturing conditions under which the target fragmentsare single stranded(iii) contacting the sample with a set of probes, wherein each probespecifically recognises a distinct target sequence within the species ofnucleic acid to be detected, wherein the target sequences are sequencesof the target fragments, and wherein each probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and target fragments, ifpresent, hybridise to the target-complementary sequence of the probes,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tail sequence(v) providing conditions for ligation so that, if a target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment, and(vi) detecting a cumulative signal which is a combination of individualsignals from all the products,

wherein detection of the signal indicates the presence of the species ofnucleic acid in the sample.

The species of nucleic acid may be quantified by a method comprising

(i) providing a sample in which the species of nucleic acid isfragmented into target fragments

(ii) providing denaturing conditions under which the target fragmentsare single stranded

(iii) contacting the sample with a set of probes, wherein each probespecifically recognises a distinct target fragment of the species ofnucleic acid to be quantified, wherein each probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and target fragments, ifpresent, hybridise to the target-complementary sequence of the probes,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tail sequence

(v) providing conditions for ligation so that, if a target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment,

(vi) detecting a cumulative signal which is a combination of individualsignals from all ligation products, and

(vii) quantifying the cumulative signal to determine a signal level,wherein the signal level is proportional to the quantity of the speciesof nucleic acid in the sample, and

thereby determining the quantity of the species of nucleic acid in thesample.

The method may be used to quantify a first species of nucleic acidrelative to a second species of nucleic acid in a sample. The method maycomprise:

(i) providing a sample in which the first and second species of nucleicacid are fragmented into target fragments

(ii) providing denaturing conditions under which the target fragmentsare single stranded

(iii) contacting the sample with a first set of probes and a second setof probes, wherein the probes of the first set specifically recognisedistinct target fragments of the first species of nucleic acid andwherein probes of the second set specifically recognise distinct targetfragments of the second species of nucleic acid, wherein each probecomprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and target fragments, ifpresent, hybridise to the target-complementary sequence of the probes,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tail sequence

(v) providing conditions for ligation so that, if a target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment,

(vi) detecting a first cumulative signal which is a combination ofindividual signals from the ligation products generated by probes of thefirst set, and quantifying it to determine a first signal level, whereinthe first signal level is proportional to the quantity of the firstspecies of nucleic acid in the sample,

(vii) detecting a second cumulative signal which is a combination ofindividual signals from the ligation products generated by probes of thesecond set, and quantifying it to determine a second signal level,wherein the second signal level is proportional to the quantity of thesecond species of nucleic acid in the sample, and

(viii) comparing the first and second signal levels, thereby determiningthe relative quantities of the first and second nucleic acid species inthe sample.

Generally, the number of probes will be at least ten for each species ofnucleic acid to be detected or quantified. The number of course refersto the number of different probes, rather than the absolute number ofmolecules of the probe. Accordingly, the nucleic acid will contain atleast ten different specific target sequences, and the cumulative signalis a combination of individual signals of at least ten unique probes,this cumulative signal representing the one species of nucleic acid.High levels of multiplex can be used to obtain correspondingly highlevels of signal amplification. For example, at least 100, at least1,000, at least 10,000 or even greater numbers of probes may be used foreach species of nucleic acid to be detected or quantified.

The method may comprise contacting a sample of fragmented chromosomeswith multiple sets of probes for binding multiple fragments of two ormore chromosomes, comprising:

a first set of probes for binding a plurality of target fragmentsspecific to a first chromosome, and

a second set of probes for binding a plurality of target fragmentsspecific to a second chromosome, and optionally

one or more further sets of probes for binding a plurality of targetfragments specific to one or more further chromosomes.

Probes within a set can share a custom sequence which is common to thatset and differs from the custom sequences of probes in other sets,allowing the probes from each set to be conveniently identified. Eachset of probes may contain at least 500, 600, 700, 800, 900 or at least1,000 different probes for binding a plurality of target fragmentsspecific to the chromosome. For example, a method may use 1,000different targeting oligonucleotides to each of chromosomes 21, 13 and18, respectively, and three different backbone oligonucleotides, one foreach chromosome subset.

It is possible to determine the relative quantities of the two or morechromosomes in a sample by detecting the products of double ligation foreach set of probes and detecting the relative quantities of the customsequences in said products.

Using probes where the targeting oligonucleotide and the upstream anddownstream oligonucleotides form a circle, motifs encoding specificalleles and or loci can be incorporated in the custom sequence in highmultiplex.

Digital Karyotyping and Non-Invasive Pre-Natal Diagnostics

Some embodiments of the the present method can provides particularadvantages in fields where precise quantification of target DNA issought. This includes a number of nucleic acid based diagnostictechniques. One such area is the analysis of cancer DNA in a biologicalsample (e.g., blood) from a patient. Another such area is non-invasivepre-natal diagnostics by analysis of cell free DNA (NIPT).

A challenge with NIPT is that a large number of specific genomefragments must be counted in order to achieve the statistical confidencerequired to diagnose an abnormality chromosomal aneuploidies (chromosomecopy number differences). Since the foetal DNA is mixed with thematernal DNA, making up 4-30% of the genetic material in a pregnantwoman's bloodstream, observing a chromosomal aneuploidy in the foetalDNA requires a very precise measurement.

The probes described herein may be used for analysing free circularisingfoetal DNA in samples of maternal blood. By using a plurality of probesdirected to different fragments of one chromosome and a plurality ofprobes directed to different fragments of a second chromosome, theprobes enable an imbalance in the relative number of the two chromosomesin the sample to be determined with high confidence. This allowschromosomal aneuploidies such as trisomy to be diagnosed from foetal DNAeven against the high background of the maternal DNA.

Probes described herein may be used for testing maternal blood samplesfrom pregnant women to detect foetal nucleic acid for the diagnosis ofchromosomal abnormalities such as trisomy, testing patient samples fortumour DNA for the diagnosis or monitoring of the presence of a tumourin the patient. Other uses include testing samples of material for thepresence of microbial nucleic acid, where detection of the microbialnucleic acid indicates infection of the material by the microbe, whichmay be an infectious agent such as a bacterium, virus or fungus. Thesample may be a tissue or blood sample from a patient.

More generally, by using hundreds or thousands of different probes, thepresent method can achieve high precision by detecting hundreds orthousands of specific nucleic acid fragments, providing advantagesacross a range of diagnostic applications. Detecting a multitude of DNAfragments from the chromosome or chromosomal loci associated with aparticular disease enables the amount of that chromosome or locus to bemeasured relative to a control chromosome or locus, so that even slightdifferences in a sample can be confidently detected.

By analysing short target fragments a large proportion of the highlyfragmented cell free DNA in maternal blood can be analysed with highefficiency. This is important since very low amounts of cell free DNAare available in maternal blood.

A method of quantifying a first chromosome or chromosomal locus relativeto a second chromosome or chromosomal locus in a sample of nucleic acidobtained from an individual may comprise

(i) providing a sample in which the first and second chromosomes orchromosomal loci are fragmented into target fragments

(ii) providing denaturing conditions under which the target fragmentsare single stranded

(iii) contacting the sample with a first set of probes and a second setof probes, wherein the probes of the first set specifically recognisedistinct target fragments of the first chromosome and wherein probes ofthe second set specifically recognise distinct target fragments of thesecond chromosome, wherein each probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and target fragments, ifpresent, hybridise to the target-complementary sequence of the probes,thereby positioning the ends of the target fragment in juxtapositionwith the 5′ end of the head sequence and the 3′ end of the tail sequence

(v) providing conditions for ligation so that, if a target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment,

(vi) detecting a first cumulative signal which is a combination ofindividual signals from the ligation products generated by probes of thefirst set, and quantifying it to determine a first signal level, whereinthe first signal level is proportional to the quantity of the firstchromosome or chromosomal locus in the sample,

(vii) detecting a second cumulative signal which is a combination ofindividual signals from the ligation products generated by probes of thesecond set, and quantifying it to determine a second signal level,wherein the second signal level is proportional to the quantity of thesecond chromosome or chromosomal locus in the sample, and

(viii) comparing the first and second signal levels, thereby determiningthe relative quantities of the first and second chromosomes orchromosomal loci in the sample.

The method may be used for diagnosing aneuploidy (e.g. trisomy) in afoetus, where the sample of nucleic acid is a sample obtained frommaternal blood and contains cell free foetal DNA mixed with maternalDNA, and wherein an unequal ratio of the first and second signal levelsis indicative of aneuploidy (e.g. trisomy).

Probes

Further aspects include probes suitable for use in the present method.Examples of probes and their features have already been described above.Some further features and examples are described here.

The probe nucleic acid is preferably DNA. However, it may be anothernucleic acid, naturally occurring or not. The standard bases of DNA areA, T, C and G, but probe nucleic acid may optionally includenon-standard nucleotides.

In general, a probe according to the present invention comprises atargeting oligonucleotide and head and tail sequences. The head and tailsequences may be part of the targeting oligonucleotide, or one or bothof them may be on a different nucleic acid molecule. Optionally, theprobe comprises the targeting oligonucleotide, a backboneoligonucleotide comprising the head sequence and a backboneoligonucleotide comprising the tail sequence. A probe therefore maycomprises one, two or three nucleic acid molecules in its non-ligatedform.

The targeting oligonucleotide is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide. The headand tail sequences have free 5′ and 3′ ends respectively, and arecomplementary to the upstream and downstream flanking sequencesrespectively. Under annealing conditions in the presence of the targetfragment, the head and tail sequences hybridise to the flankingsequences, defining a gap between the 5′ end of the head sequence andthe 3′ end of the tail sequence, wherein the target fragment hybridisesto the target-complementary sequence in the gap, thereby positioning theends of the target fragment in juxtaposition with the 5′ end of the headsequence and the 3′ end of the tail sequences.

The probes may be designed so that hybridisation of the target fragmentin the gap completes a circle of nucleic acid, the circle comprising thetarget fragment and the head and tail sequences.

The head and/or tail sequence of the probe is preferably joined to acustom sequence which is not complementary to other regions of the probeor to the target fragment.

In some embodiments of the probe, a single nucleic acid moleculecomprises the head and tail sequences.

The head and tail sequences may be separate from the targetingoligonucleotide so that they bind in trans to the flanking sequences.For example, the head and tail sequences may be at 5′ and 3′ endsrespectively of a backbone oligonucleotide. A custom sequence can beincluded between the head and tail sequences of the backboneoligonucleotide. An example of such a probe is shown in FIG. 1 and FIG.3. Alternatively, the head and tail sequences of the backboneoligonucleotide may be adjacent, with no intervening nucleotidesequence. In such a case, the flanking sequences of the targetingoligonucleotide hybridise along the full length of the backboneoligonucleotide and may circularise it.

The probes may be designed so that the head sequence is a 5′ end of thetargeting oligonucleotide and/or the tail sequence is a 3′ end of thetargeting oligonucleotide, so that hybridisation of the target fragmentin the gap completes a strand of nucleic acid comprising the targetfragment, the head and tail sequences, the target complementary sequenceand the flanking sequences. The head and tail sequences may be at endsof the targeting oligonucleotide and bind in cis to the flankingsequences. An example of such a probe is shown in FIG. 4. In thisversion of the probe, the head and tail sequences and the targetcomplementary sequence all become circularised with the target fragment.Custom sequences can be positioned in the loops of the oligonucleotide.The probe nucleic acid is relatively long but has the advantage ofjoining the oligonucleotide structure into one molecule that ispre-assembled and does not require hybridisation of different probenucleic acid molecules.

Probes can also be designed with a backbone oligonucleotide, which is aseparate molecule of nucleic acid from the targeting oligonucleotide.The tail sequence can be a 3′ end of the targeting oligonucleotide andthe head sequence a 5′ end of a backbone oligonucleotide. Alternativelythe head sequence can be a 5′ end of the targeting oligonucleotide andthe tail sequence a 3′ end of a backbone oligonucleotide. A customsequence can be introduced in the targeting oligonucleotide, for exampleto provide a loop between the head or tail sequence and the flankingsequence. An advantage with using this probe approach is that adetection sequence can be introduced in the loop and is associated withthe target complementary sequence, which can be advantageous formultiplex methods, especially higher multiplexes with high-plexdetection schemes. The backbone oligonucleotide can further comprise acustom sequence. By providing the probe in two oligonucleotides, theprobe nucleic acid molecules are shorter than the single oligonucleotideversion but maintain the same function.

Another design of the probe provides the head and tail sequences on twobackbone oligonucleotides. Thus, the probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide,

a backbone oligonucleotide comprising a head sequence having a free 5′end, and

a backbone oligonucleotide comprising a tail sequence having a free 3′end,

wherein the head and tail oligonucleotide sequences are complementary tothe upstream and downstream flanking sequences respectively.

One backbone oligonucleotide may carry a capture moiety, in which casethe other backbone oligonucleotide is used for detection and may carry aheterogeneous label. One or both backbone oligonucleotides may furthercomprise a custom sequence. Alternatively or additionally, the targetingoligonucleotide may include a custom sequence.

Under annealing conditions in the presence of the target fragment, thehead and tail sequences hybridise to the flanking sequences, defining agap between the 5′ end of the head sequence and the 3′ end of the tailsequence, wherein the target fragment hybridises to thetarget-complementary sequence in the gap, thereby positioning the endsof the target fragment in juxtaposition with the 5′ end of the headsequence and the 3′ end of the tail sequences. Hybridisation of thetarget fragment in the gap completes a strand of nucleic acid comprisingthe target fragment and the head and tail sequences. The strand carriesthe capture moiety and the label, permitting detection using thecapture/detect methods described elsewhere herein.

Kits and Sets of Probes

A further aspect of this disclosure is set of probes for binding singlestranded target nucleic acid fragments, comprising a plurality ofprobes, the probes having a plurality of different target-complementarysequences for the binding multiple different target fragments.

The set of probes may be for binding multiple fragments of a humanchromosome, wherein each probe in the set is for binding a differenttarget fragment specific to that chromosome. Such probes may all includea common custom sequence, as part of the targeting oligonucleotide or aspart of a backbone oligonucleotide.

Multiple sets of probes can be provided for binding different fragmentsof two or more human chromosomes, comprising:

a first set of probes for binding a plurality of target fragmentsspecific to a first chromosome, and

a second set of probes for binding a plurality of target fragmentsspecific to a second chromosome, and optionally

one or more further sets of probes for binding a plurality of targetfragments specific to one or more further chromosomes. The probes withina set can share a custom sequence which is common to that set anddiffers from the custom sequences of probes in other sets.

Kits can also be provided, comprising sets of probes in solution in oneor more containers.

Uses

The probes, sets of probes and kits described herein may be used fortesting samples for the presence of target nucleic acid fragments. Theymay be used for identifying the presence of a defined target fragment ina sample of fragmented nucleic acid in vitro.

One aspect includes the use of a probe for testing a sample for thepresence of a target single stranded nucleic acid fragment,

wherein the probe comprises a targeting oligonucleotide containing asequence which is the exact complement of the target fragment, and headand tail oligonucleotide sequences which hybridise adjacent to thetarget fragment on the targeting oligonucleotide,

wherein hybridisation between the target fragment and the probetemplates the target fragment for ligation to the head and tailsequences.

Examples of such probes and of their use in methods of testing samplesare described in more detail elsewhere herein. Uses include testingmaternal blood samples from pregnant women to detect foetal nucleic acidfor the diagnosis of chromosomal abnormalities such as trisomy, andtesting patient samples for tumour DNA for the diagnosis or monitoringof the presence of a tumour in the patient. Other uses include testingsamples of material for the presence of microbial nucleic acid, wheredetection of the microbial nucleic acid indicates infection of thematerial by the microbe, which may be an infectious agent such as abacterium, virus or fungus. The sample may be a tissue or blood samplefrom a patient.

EXAMPLES

The following example is provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof.

Example 1

A protocol suitable for performing the method illustrated in FIG. 1 isas follows: 1) 10 ng of DNA is digested with 1 unit of restrictionenzyme in corresponding compatible restriction enzyme buffer. Thereaction is incubated in 37 C for 1 h, followed by enzymaticdeactivation at 80 C for 20 min. 2) The DNA fragments are denatured tosingle stranded fragments at 95 C for 10 min and mixed with probes andligase to form circles. The probe pool are added in 10 pM individualconcentration along with 1 U of Ampligase (Epicentre) and incubated at55 C for 1 h in ligase buffer. 3) 1 U Exonuclease is added to removenon-reacted probes and fragments. I U of Lambda exonuclease (Epicentre)is added at 37 C for 1 h in corresponding exonuclease buffer followed byenzyme inactivation at 80 C for 20 min. 4) The remaining circles areamplified by RCA. 1 U of phi29 polymerase (New England Biolabs) is addedin corresponding phi29 buffer and nucleotides (dNTPs) at 37 C for 1 h.

Example 2

A protocol suitable for performing the method illustrated in FIG. 2 isas follows: 1) 10 ng of DNA is digested with 1 unit of restrictionenzyme in corresponding compatible restriction enzyme buffer. Thereaction is incubated in 37 C for 1 h, followed by enzymaticdeactivation at 80 C for 20 min. 2) The DNA fragments are denatured tosingle stranded fragments at 95 C for 10 min and mixed with probes andligase to form linear ligation products. The probe pool are added in 10pM individual concentration along with 1 U of Ampligase (Epicentre) andincubated at 55 C for 1 h in ligase buffer. 3) The ligation product iscaptured on magnetic streptavidin beads. To remove non-reacted probesand fragments, the solution is mixed with 10 ml M-280 streptavidincoated magnetic beads (Invitrogen) in Tris-HCl (pH 7.5), 3.5 mM EDTA and0.07% Tween-20 in a final volume of 200 ml, and incubated at roomtemperature for 15 min. After incubation, the beads are collected usinga ring magnet and supernatant removed.

Example 3 Materials and Methods

Sample Preparation:

10 ml blood was collected from each subject into a cell-free DNA tube(Streck, Omaha, Nebr.). Plasma was isolated from blood by a doublecentrifugation protocol (1600 g for 10 min, followed by 16 000 g for 10min, after a tube transfer following the first spin). cfDNA was isolatedby the Qiagen ccf nucleic acid kit (Qiagen, Hilden, Germany) accordingto the manufacturer's protocol. The resulting DNA was eluted in 50 ul ofbuffer (part of the Qiagen kit).

Probe and Backbone Design:

The multiplexed probe technology herein described enables specific andsimultaneous amplification of thousands of chromosomal fragments. Probeswere designed to capture 2500-5000 fragments (targets) from each ofchromosomes 21, 18, and 13. Targets were selected to have uniquesequence in the genome, uniformed AT/GC composition, not include knownpolymorphism nor CNVs in target sequence, and a size between 18-35 bp.Probes targeting 2500 fragments from each chromosome 13 and 18 werepooled together with 5000 probes targeting fragments from chromosome 21to create a single oligo probe pool.

Example sequence of probes, “N” represents target complementarysequence:

(SEQ ID NO: 1) ATGTGACCCTTCCGTCTGTTGAGTTAGGCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCGTGCCTTGTCATTCGGGAGCACTAACTGCTG

The backbones, with head and tail sequences complementary to the ends ofthe probe, were designed to include sequence motifs for both sequencingand digital counting. Two backbones were used in the experimentsoutlined in the result section; one complementary to probes targetingchromosome 13 and 18:

(/5Phos/CGCACACGATTAAGGTCCAGTCACAGGCAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTNNNNNNNNNNGTGTAGATCTCGGTGGTCGCCGTATCATTTCATGCTGCTAACGGTCGAGTCGGACAGGTGGCTCCACTAAATAGACGCA); SEQ ID NO:2, and one backbonetargeting chromosome 21:

(/5Phos/GGCCTAACTCAACAGACGGAAGGGTCACATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTNNNNNNNNNNGTGTAGATCTCGGTGGTCGCCGTATCATTTCATGCTGCTAACGGTCGAGCAGTTAGTGCTCCCGAATGACAAGGCACGA; SEQ ID NO: 3).

Biochemistry Probe Protocol:

50 ul of purified cfDNA was digested with 5 U of MseI (New EnglandBiolabs) in 1×NEB4 buffer (New England Biolabs) and 1×BSA in a totalvolume of 55 ul at 37 C in 30 min followed by heat inactivation at 65 Cin 20 min. The digested DNA was then mix with ligation mix along withprobes and backbones. The 55 ul of digested DNA was mixed with probes (1pM/probe), backbones (60 nM each), 1× ligation buffer (Epicentre), 100 Uof Ampligase (Epicentre), 1 mM NAD, and 5 mM Mg²⁺ to a total volume of70 ul. The digested fragments were first denatured to single strandedDNA at 95 C in 5 min followed by 55 C hybridization and ligation in 16h. The ligation mix was then treated with exonucleases to remove anyremaining linear DNA molecules. The ligation reaction was mixed with 20U of ExoI (NEB) and 5 U of ExoIII (NEB) and 1×BSA tot total volume of 75ul at 37 C for 60 min followed by heat inactivation at 65 C for 10 min.

Analysis:

For sequencing analysis, the exo treated circles was amplified withsequencing primers complementary to the Illumina sequencing instrumentand subsequently loaded on the Illumina Miseq instrument according tomanufacturers protocol.

For digital analysis, the exo treated reactions was subjected to arolling circle amplification reaction (RCA) to generate discrete DNAobjects of concatemeric copies of the circle. 37.5 ul of exo treatedcircles were mixed with 4 mM DTT, 3 U of phi29 polymerase (NEB), 0.1 uMprimer, 1 mM dNTP mix (NEB) and 1×BSA in a total volume in 50 ul, andincubated at 37 C for 1 h followed by a heat inactivation at 65 C for 10min. The RCA reaction was then labeled with fluorescently labeledoligonucleotides complementary to the backbone sequence. 50 ul of RCAproducts was mixed with 0.1% Tween 20 (Sigma), 5 nM labeledoligonucleotides, and 2×SSC (Sigma) in a total volume of 100 ul. Thelabeled RCA-products were finally deposited on a microscope slide coatedwith Poly-lysine (Sigma) and counted in a fluorescent microscope.

Results

The probe method herein described was demonstrated on Illuminasequencing and a digital counting system. To demonstrate the performanceof the probe method, a DNA sample with trisomy 21 was mixed with DNAextracted from normal plasma samples (3-5 ml plasma) in differentconcentrations. The samples was then carried through the probe methodand evaluated by sequencing.

For the results shown in FIG. 8, 100 ng of cell line DNA was subjectedto the protocol described above. 10,000 probes were mixed in a pool tospecifically circularize 10,000 corresponding chromosomal fragments fromchromosome 13, 18, and 21. The 10,000 resulting circles were thenamplified with Illumina-corresponding PCR primers and analyzed on gelprior sequencing. Lane 1 corresponds to DNA ladder, lane 2 the DNAsample after digestion, and lane 3 the PCR product with 10,000 amplifiedfragments.

For the results shown in FIG. 9, 12 normal plasma samples were analyzedin parallel with samples carry DNA with trisomy 21 in differentconcentrations. DNA were extracted and processed through the 10K-plexprobe protocol and finally sequenced on Illumina sequencer. Using aconfidence interval providing 99% specificity, the positive samples aredetected with a 90% sensitivity based on the estimated normaldistributions.

To demonstrate the principle of converting targeted fragments to labeledDNA objects, 10% of DNA with trisomy 21 was added to 20 ng normal cellline DNA and carried through the probe method. The resulting labeledRCA-products were randomly deposited on a microscope slide and counted.Probes targeting fragments derived from chromosome 21 was labeled withone color and fragments derived from Chr. 13 and 18 with a referencecolor. These results are shown in FIG. 10. Panel (A) of FIG. 10 shows animage from a microscope, showing labeled and detected RCA-products. Bylabeling all fragments from chromosome 13 with one fluorophore andfragments from a reference chromosome with a second fluorophore, a ratiomeasurement can be achieve. Panel (B): 20 ng of DNA processed throughthe 10K-plex probe protocol and converted to labeled RCA-products. TheRCA-products were analyzed in parallel with samples carry a 10% additionof trisomy 21 DNA. 12 normal DNA samples (sample #1-12) were analyzed inparallel with three positive samples (sample #13-15).

FURTHER DESCRIPTION

The following clauses are part of the description.

1. A method of testing a sample for the presence of a target nucleicacid fragment, comprising(i) providing a sample of fragmented nucleic acid(ii) providing denaturing conditions under which the target fragment issingle stranded(iii) contacting the sample with a nucleic acid probe comprising

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively,

(iv) providing annealing conditions under which the head and tailsequences hybridise to the flanking sequences, and the target fragment,if present, hybridises to the target-complementary sequence, therebypositioning the ends of the target fragment in juxtaposition with the 5′end of the head sequence and the 3′ end of the tail sequence(v) providing conditions for ligation so that, if the target fragment ispresent, the 3′ end of the target fragment is ligated to the 5′ end ofthe head sequence to form a first ligation junction, and the 5′ end ofthe target fragment is ligated to the 3′ end of the tail sequence toform a second ligation junction, producing a product of double ligationcomprising a continuous strand of nucleic acid comprising the head andtail sequences and the target fragment, and(vi) detecting whether the product of double ligation is present,

wherein detecting the product of double ligation indicates the presenceof the target fragment in the sample.

2. A method according to clause 1, wherein the sample of fragmentednucleic acid is a restriction enzyme digest and the target fragment is arestriction fragment.3. A method according to clause 1 or clause 2, wherein the 5′ end of thehead sequence and the 3′ end of the target fragment hybridise toadjacent nucleotides of the targeting oligonucleotide, and the 3′ end ofthe tail sequence and the 5′ end of the target fragment hybridise toadjacent nucleotides of the targeting oligonucleotide.4. A method according to any of the preceding clauses, wherein the stepof detecting the product of double ligation comprises providingconditions for amplification across the first and second ligationjunctions of the continuous strand of nucleic acid, and detectingwhether an amplification product is present.5. A method according to any of the preceding clauses, wherein thecontinuous strand of nucleic acid comprising the head and tail sequencesand the target fragment is a circle of nucleic acid.6. A method according to clause 5, wherein the step of detecting theproduct of double ligation comprises providing conditions for rollingcircle replication and detecting whether a product of rolling circlereplication is present.7. A method according to clause 6, wherein the rolling circlereplication is hyper branched rolling circle replication.8. A method according to any of clauses 5 to 7, wherein the probecomprises the head and tail sequences on one nucleic acid molecule.9. A method according to clause 8, wherein the probe comprises abackbone oligonucleotide having the head and tail sequences at its 5′end 3′ ends respectively, wherein the head and tail sequences of thebackbone oligonucleotide bind in trans to the flanking sequences of thetargeting oligonucleotide under the annealing conditions.10. A method according to clause 9, wherein the backbone oligonucleotidecomprises a custom sequence between the head and tail sequences, whereinthe custom sequence is not complementary to other regions of the probeor to the target fragment.11. A method according to clause 9, wherein the head and tail sequencesof the backbone oligonucleotide are adjacent.12. A method according to any of clauses 5 to 8, wherein the head andtail sequences are at ends of the targeting oligonucleotide and bind incis to the flanking sequences under the annealing conditions.13. A method according to clause 12, wherein the targetingoligonucleotide comprises a custom sequence between the targetingoligonucleotide and the head and/or tail sequence, wherein the customsequence is not complementary to other regions of the probe or to thetarget fragment.14. A method according to any of clauses 1 to 7, wherein the tailsequence is at the 3′ end of the targeting oligonucleotide, and theprobe comprises a backbone oligonucleotide having the head sequence atits 5′ end,

wherein under the annealing conditions the tail sequence binds in cis tothe downstream flanking sequence of the targeting oligonucleotide, andthe head sequence of the backbone oligonucleotide binds in trans to theupstream flanking sequence of the targeting oligonucleotide.

15. A method according to clause 14, wherein the backboneoligonucleotide comprises a pair of inverted repeat sequences, wherein

under the annealing conditions the inverted repeat sequences form ahairpin structure, thereby positioning the 3′ end of the backboneoligonucleotide in juxtaposition with the 5′ end of the targetingoligonucleotide, and wherein

under the conditions for ligation, the 5′ end of the targetingoligonucleotide is ligated to the 3′ end of the backboneoligonucleotide, so that the product of double ligation is a circle ofnucleic acid comprising the targeting oligonucleotide, the targetfragment and the backbone oligonucleotide.

16. A method according to any of clauses 1 to 7, wherein the headsequence is at the 5′ end of the targeting oligonucleotide, and theprobe comprises a backbone oligonucleotide having the tail sequence atits 3′ end,

wherein under the annealing conditions the head sequence binds in cis tothe upstream flanking sequence of the targeting oligonucleotide, and thetail sequence of the backbone oligonucleotide binds in trans to thedownstream flanking sequence of the targeting oligonucleotide.

17. A method according to clause 16, wherein the backboneoligonucleotide comprises a pair of inverted repeat sequences, wherein

under the annealing conditions the inverted repeat sequences form ahairpin structure, thereby positioning the 5′ end of the backboneoligonucleotide in juxtaposition with the 3′ end of the targetingoligonucleotide, and wherein

under the conditions for ligation, the 3′ end of the targetingoligonucleotide is ligated to the 5′ end of the backboneoligonucleotide, so that the product of double ligation is a circle ofnucleic acid comprising the targeting oligonucleotide, the targetfragment and the backbone oligonucleotide.

18. A method according to any of clauses 14 to 17, wherein the backboneoligonucleotide comprises a custom sequence between the inverted repeatsequence, so that under the annealing conditions the backboneoligonucleotide forms a hairpin loop.19. A method according to any of clauses 1 to 4, wherein the continuousstrand of nucleic acid comprising the head and tail sequences and thetarget fragment is a linear strand of nucleic acid.20. A method according to clause 19, wherein the tail sequence is at the3′ end of the targeting oligonucleotide, and the probe comprises abackbone oligonucleotide having the head sequence at its 5′ end,

wherein under the annealing conditions the tail sequence binds in cis tothe downstream flanking sequence of the targeting oligonucleotide, andthe head sequence of the backbone oligonucleotide binds in trans to theupstream flanking sequence of the targeting oligonucleotide.

21. A method according to any of clauses 14, 15 or 20, wherein thetargeting oligonucleotide comprises a custom sequence between thedownstream flanking sequence and the tail sequence, so that under theannealing conditions the targeting oligonucleotide forms a hairpin loop.22. A method according clause 19, wherein the head sequence is at the 5′end of the targeting oligonucleotide, and the probe comprises a backboneoligonucleotide having the tail sequence at its 3′ end,

wherein under the annealing conditions the head sequence binds in cis tothe upstream flanking sequence of the targeting oligonucleotide, and thetail sequence of the backbone oligonucleotide binds in trans to thedownstream flanking sequence of the targeting oligonucleotide.

23. A method according to any of clauses 16, 17 or 22, wherein thetargeting oligonucleotide comprises a custom sequence between the headsequence and the upstream flanking sequence, so that under the annealingconditions the targeting oligonucleotide forms a hairpin loop.24. A method according to any of clauses 14 to 18 or 20 to 23, whereinthe backbone oligonucleotide carries a capture moiety.25. A method according to clause 19, wherein the probe comprises abackbone oligonucleotide comprising a head sequence having a free 5′end, and a backbone oligonucleotide comprising a tail sequence having afree 3′ end, wherein under the annealing conditions the head and tailsequences bind in trans to the flanking sequences of the targetingoligonucleotide.26. A method according to clause 25, wherein one or both backboneoligonucleotides further comprise a custom sequence, wherein the customsequence is not complementary to other regions of the probe or to thetarget fragment.27. A method according to clause 25 or clause 26, wherein one of thebackbone oligonucleotides carries a capture moiety.28. A method according to clause 27, wherein the other backboneoligonucleotide carries a heterogeneous label.29. A method according to clause 28, wherein the label is a fluorophore.30. A method according to clause 24 or any of clauses 27 to 29, whereinthe step of detecting whether the product of double ligation is presentcomprises capturing the backbone oligonucleotide on a substrate via thecapture moiety, washing the substrate to remove unligated probes andretaining a captured fraction comprising the substrate and capturedbackbone oligonucleotide, and testing for the presence of the product ofdouble ligation in the captured fraction.31. A method according to clause 28 or clause 29, wherein the step ofdetecting whether the product of double ligation is present comprisescapturing the backbone oligonucleotide on a substrate via the capturemoiety, washing the substrate to remove unligated probes and retaining acaptured fraction comprising the substrate and captured backboneoligonucleotide, and testing for the presence of the label in thecaptured fraction.32. A method according to clause 24 or any of clauses 27 to 31, whereinthe capture moiety is biotin.33. A method according to any of the preceding clauses, wherein thetarget-complementary sequence has a length of 10 to 30 nucleotides.34. A method according to any of the preceding clauses, wherein thetarget-complementary sequence has fewer than 5 base pair mismatches withthe target fragment.35. A method according to clause 34, wherein the target-complementarysequence is the exact complement of the target fragment.36. A method according to any of the preceding clauses, wherein theflanking sequences each have a length of 10 to 30 nucleotides.37. A method according to any of the preceding clauses, wherein theupstream and downstream flanking sequences are different from eachother.38. A method according to any of the preceding clauses, wherein the headsequence has fewer than 5 base pair mismatches with the upstreamflanking sequence and the tail sequence has fewer than 5 base pairmismatches with the downstream flanking sequence.39. A method according to clause 38, wherein the head sequence is theexact complement of the upstream flanking sequence and the tail sequenceis the exact complement of the downstream flanking sequence.40. A method according to any of the preceding clauses, wherein thetargeting oligonucleotide is linear.41. A method according to any of the preceding clauses, wherein thesample is a sample of fragmented human chromosomes and the targetfragment is a human genome fragment specific to one chromosome.42. A method according to clause 41, wherein the target fragment isspecific to one locus of the human genome.43. A method according to any of the preceding clauses, wherein theprobe nucleic acid is DNA.44. A method according to any of the preceding clauses, wherein themethod comprises multiplex testing for multiple different target nucleicacid fragments using a plurality of the probes in parallel.45. A method according to clause 44, wherein the method comprisescontacting a sample of fragmented chromosomes with a set of probes forbinding multiple fragments of a chromosome, wherein each probe in theset is for binding a different target fragment specific to thatchromosome.46. A method according to clause 45, wherein the probes share a commoncustom sequence.47. A method according to clause 44, wherein the method comprisescontacting a sample of fragmented chromosomes with sets of probes forbinding multiple fragments of two or more chromosomes, wherein the setsof probes comprise:

a first set of probes for binding a plurality of target fragmentsspecific to a first chromosome, and

a second set of probes for binding a plurality of target fragmentsspecific to a second chromosome, and optionally

one or more further sets of probes for binding a plurality of targetfragments specific to one or more further chromosomes.

48. A method according to clause 47, wherein each set of probescomprises at least 500 different probes for binding a plurality oftarget fragments specific to the chromosome.49. A method according to clause 47 or clause 48, wherein the probeswithin a set share a custom sequence which is common to that subset anddiffers from the custom sequences of probes in other sets.50. A method according to clause 49, comprising determining the relativequantities of the two or more chromosomes in the sample by detecting theproducts of double ligation for each set of probes and detecting therelative quantities of the custom sequences in said products.51. A method according to any of clauses 45 to 50, wherein thechromosome or chromosomes are human.52. A nucleic acid probe for binding a single stranded target nucleicacid fragment, wherein the probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail sequences are complementary to the upstream anddownstream flanking sequences respectively

so that under annealing conditions in the presence of the targetfragment, the head and tail sequences hybridise to the flankingsequences, defining a gap between the 5′ end of the head sequence andthe 3′ end of the tail sequence, wherein the target fragment hybridisesto the target-complementary sequence in the gap, thereby positioning theends of the target fragment in juxtaposition with the 5′ end of the headsequence and the 3′ end of the tail sequences, and wherein

hybridisation of the target fragment in the gap completes a circle ofnucleic acid, the circle comprising the target fragment and the head andtail sequences.

53. A nucleic acid probe according to clause 52, wherein the head and/ortail sequence is joined to a custom sequence, wherein the customsequence is not complementary to other regions of the probe or to thetarget fragment.54. A nucleic acid probe according to clause 52 or clause 53, wherein asingle nucleic acid molecule comprises the head and tail sequences.55. A probe according to clause 52 or clause 53, wherein the head andtail sequences are separate from the targeting oligonucleotide and bindin trans to the flanking sequences.56. A probe according to clause 55, wherein the head and tail sequencesare at 5′ and 3′ ends respectively of a backbone oligonucleotide.57. A probe according to clause 56, wherein the backbone oligonucleotidecomprises a custom sequence between the head and tail sequences, whereinthe custom sequence is not complementary to other regions of the probeor to the target fragment.58. A probe according to clause 56, wherein the head and tail sequencesof the backbone oligonucleotide are adjacent.59. A nucleic acid probe for binding a single stranded target nucleicacid fragment, wherein the probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide, and

head and tail sequences having free 5′ and 3′ ends respectively, whereinthe head and tail oligonucleotide sequences are complementary to theupstream and downstream flanking sequences respectively

so that under annealing conditions in the presence of the targetfragment, the head and tail sequences hybridise to the flankingsequences, defining a gap between the 5′ end of the head sequence andthe 3′ end of the tail sequence, wherein the target fragment hybridisesto the target-complementary sequence in the gap, thereby positioning theends of the target fragment in juxtaposition with the 5′ end of the headsequence and the 3′ end of the tail sequences, and wherein

the head sequence is a 5′ end of the targeting oligonucleotide and/orthe tail sequence is a 3′ end of the targeting oligonucleotide, so thathybridisation of the target fragment in the gap completes a strand ofnucleic acid comprising the target fragment, the head and tailsequences, the target complementary sequence and the flanking sequences.

60. A probe according to clause 52 or clause 59, wherein the head andtail sequences are at ends of the targeting oligonucleotide and bind incis to the flanking sequences.61. A probe according to clause 52 or clause 59, wherein the tailsequence is a 3′ end of the targeting oligonucleotide and the headsequence is a 5′ end of a backbone oligonucleotide separate from thetargeting oligonucleotide.62. A probe according to clause 52 or clause 59, wherein the headsequence is a 5′ end of the targeting oligonucleotide and the tailsequence is a 3′ end of a backbone oligonucleotide separate from thetargeting oligonucleotide.63. A probe according to clause 61 or clause 62, wherein the backboneoligonucleotide further comprises a custom sequence, wherein the customsequence is not complementary to other regions of the probe or to thetarget fragment.64. A nucleic acid probe for binding a single stranded target nucleicacid fragment, wherein the probe comprises

a targeting oligonucleotide which is longer than the target fragment andcontains an internal target-complementary sequence, so thathybridisation between the targeting oligonucleotide and the targetfragment forms a double stranded sequence located between upstream anddownstream flanking sequences of the targeting oligonucleotide,

a backbone oligonucleotide comprising a head sequence having a free 5′end, and

a backbone oligonucleotide comprising a tail sequence having a free 3′end,

wherein the head and tail oligonucleotide sequences are complementary tothe upstream and downstream flanking sequences respectively, and wherein

one backbone oligonucleotide carries a capture moiety and the otherbackbone oligonucleotide carries a heterogeneous label,

so that under annealing conditions in the presence of the targetfragment, the head and tail sequences hybridise to the flankingsequences, defining a gap between the 5′ end of the head sequence andthe 3′ end of the tail sequence, wherein the target fragment hybridisesto the target-complementary sequence in the gap, thereby positioning theends of the target fragment in juxtaposition with the 5′ end of the headsequence and the 3′ end of the tail sequences, and wherein

hybridisation of the target fragment in the gap completes a strand ofnucleic acid comprising the target fragment and the head and tailsequences, wherein the strand carries the capture moiety and the label.

65. A probe according to clause 64, wherein the capture moiety isbiotin.66. A probe according to clause 64 or clause 65, wherein the label is afluorophore.67. A probe according to any of clauses 64 to 66, wherein one or bothbackbone oligonucleotides further comprise a custom sequence, whereinthe custom sequence is not complementary to other regions of the probeor to the target fragment.68. A probe according to any of clauses 52 to 67, wherein the targetingoligonucleotide further comprises a custom sequence which is notcomplementary to other regions of the probe or to the target fragment.69. A probe according to any of the preceding clauses, wherein thetarget-complementary sequence has a length of 10 to 30 nucleotides.70. A probe according to any of the preceding clauses, wherein thetarget-complementary sequence has fewer than 5 base pair mismatches withthe target fragment.71. A probe according to clause 70, wherein the target-complementarysequence is the exact complement of the target fragment.72. A probe according to any of clauses 52 to 71, wherein the flankingsequences each have a length of 10 to 30 nucleotides.73. A probe according to any of clauses 52 to 72, wherein the upstreamand downstream flanking sequences of the targeting oligonucleotide aredifferent from each other.74. A probe according to any of clauses 52 to 73, wherein the headsequence has fewer than 5 base pair mismatches with the upstreamflanking sequence and the tail sequence has fewer than 5 base pairmismatches with the downstream flanking sequence.75. A probe according to clause 74, wherein the head and tail sequencesare the exact complement of the flanking sequences.76. A probe according to any of clauses 52 to 75, wherein the targetingoligonucleotide is linear.77. A probe according to any of clauses 52 to 76, wherein the targetfragment is a restriction endonuclease fragment.78. A probe according to any of clauses 52 to 77, wherein the targetfragment is a human genome fragment.79. A probe according to clause 78, wherein the target fragment is ahuman genome fragment specific to one chromosome.80. A probe according to clause 79, wherein the target fragment isspecific to one locus of the human genome.81. A probe according to any of clauses 52 to 80, wherein the probenucleic acid is DNA.82. A set of probes for binding single stranded target nucleic acidfragments, comprising a plurality of probes according to any of clauses52 to 81, the probes having a plurality of differenttarget-complementary sequences for the binding multiple different targetfragments.83. A set of probes according to clause 82 which is for binding multiplefragments of a human chromosome, wherein each probe in the set is forbinding a different target fragment specific to that chromosome.84. A set of probes according to clause 83, wherein the probes share acommon custom sequence.85. Sets of probes for binding different fragments of two or more humanchromosomes, comprising:

a first set of probes for binding a plurality of target fragmentsspecific to a first chromosome, and

a second set of probes for binding a plurality of target fragmentsspecific to a second chromosome, and optionally

one or more further sets of probes for binding a plurality of targetfragments specific to one or more further chromosomes.

86. Sets of probes according to clause 85, wherein the probes within aset share a custom sequence which is common to that set and differs fromthe custom sequences of probes in other sets.87. A kit comprising a set or sets of probes according to any of clauses82 to 86 in solution in one or more containers.88. Use of a probe according to any clauses 52 to 81, a set of probesaccording to any of clauses 82 to 86, or a kit according to clause 87,for testing a sample for the presence of a target nucleic acid fragment.89. Use of a probe for testing a sample for the presence of a targetsingle stranded nucleic acid fragment,

wherein the probe comprises a targeting oligonucleotide containing asequence which is the exact complement of the target fragment, and headand tail oligonucleotide sequences which hybridise adjacent to thetarget fragment on the targeting oligonucleotide,

wherein hybridisation between the target fragment and the probetemplates the target fragment for ligation to the head and tailsequences.

90. Use according to clause 89, wherein the probe is as defined in anyof clauses 52 to 81.

An embodiment provides a method of processing a nucleic acid sample,comprising: a) hybridizing a sample comprising a target fragment to anucleic acid probe comprising: i. a head sequence and a tail sequence,wherein the head and tail sequences are at the ends of a firstoligonucleotide molecule; and ii. a splint sequence comprising, inorder: an upstream flanking sequence that is complementary to the headsequence; a target complementary sequence that is complementary to thetarget fragment; and a downstream flanking sequence that iscomplementary to the tail sequence; thereby producing a hybridizationproduct in which the ends of the target fragment are ligatably adjacentto the ends of the head and tail sequences in the first oligonucleotidemolecule; and b) ligating the ends of the target fragment to the ends ofthe head and tail sequences of the first oligonucleotide molecule,thereby producing a cyclic product that comprises the target fragmentand the head and tail sequences.

In any embodiment, the method may further comprise amplifying the cyclicproduct by rolling circle amplification using a primer that hybridizesto the first oligonucleotide molecule or the splint sequence. In theseembodiments, the method may further comprise quantify the number ofrolling circle amplification products produced, thereby providing anestimate of the amount of said target fragment in the sample.

In some embodiments, the splint sequence may be in the firstoligonucleotide molecule.

In some embodiments, the splint sequence may be in a secondoligonucleotide molecule.

In any embodiment, the target-complementary sequence may be 10 to 30nucleotides in length.

In any embodiment, the target-complementary sequence may contains one ormore mismatches to the target fragment.

In any embodiment, the flanking sequences may be 10 and 40 nucleotidesin length.

In any embodiment, the sample may be digested with a restriction enzyme.

In any embodiment, the sample may comprise genomic DNA, e.g., humangenomic DNA. In these embodiments, the sample may comprise cell-free DNAisolated from blood. For example, in any embodiment, the sample maycomprise cell-free DNA isolated from the bloodstream of a pregnanthuman.

In some embodiments, the splint sequence may be in a secondoligonucleotide molecule that comprises an capture moiety, e.g., abiotin moiety. In these embodiments, the method may comprise: c)immobilizing the cyclic product by binding the capture moiety to a solidphase; and d) washing the solid phase to remove unligated nucleic acidand other reaction components, thereby enriching for the cyclic product.

In any embodiment, the target fragment may be from chromosome 21, 13 or18.

In some embodiments, the method may comprise hybridizing the sample witha set of at least 50 of said probes, wherein said probes targetdifferent fragments on the same chromosome, and wherein the methodresults in a plurality of cyclic products that comprise the targetfragments.

In these embodiments, the method may comprise hybridizing the samplewith a first set and a second set of said sets of probes, wherein thefirst and second sets target a first chromosome and a second chromosome,respectively, amplifying the cyclic products by rolling circleamplification (RCA) and comparing the number of RCA productscorresponding to the first chromosome to the number of RCA productscorresponding to the first chromosome.

In these embodiments, the method may comprise hybridizing the samplewith a first set and a second set of said sets of probes, wherein thefirst and second sets target a first and second regions on a chromosome,respectively, amplifying the cyclic products by rolling circleamplification (RCA) and comparing the number of RCA productscorresponding to the first region to the number of RCA productscorresponding to the second region.

Also provided herein is a composition comprising a nucleic acid probe,as described above. In some embodiments, the nucleic acid probe maycomprise: i. a head sequence and a tail sequence, wherein the head andtail sequences are at opposite ends of a first oligonucleotide molecule;and ii. a splint sequence comprising, in order: an upstream flankingsequence that is complementary to the head sequence; a targetcomplementary sequence that is complementary to a target fragment in thehuman genome; and a downstream flanking sequence that is complementaryto the tail sequence; wherein the probe is designed so that, when thefirst oligonucleotide, the splint sequence, and the target fragment arehybridized to one another, the ends of the target fragment are ligatablyadjacent to the ends of the head and tail sequences in the firstoligonucleotide molecule.

In any composition embodiment, the composition may comprise a first setof at least 50 of the nucleic acid probes, wherein the targetcomplementary sequences of said probes are complementary to differenttarget fragments of a first human chromosome, e.g., human chromosome is21, 13 or 18. In these embodiments, the composition may optionallycomprise a second set of at least 50 of said nucleic acid probes,wherein the target complementary sequences of said probes of the secondset are complementary to different target fragments of a second humanchromosome, e.g., chromosomes 13 or 18 (if the first chromosome ischromosome 21).

1-73. (canceled)
 74. A kit comprising: a first set of at least 1,000targeting oligonucleotides, each comprising: (i) an internaltarget-complementary sequence that is in the range of 10 to 100nucleotides in length and complementary to a sequence in humanchromosome 21, wherein the different members of the first set havedifferent internal target-complementary sequences, (ii) an upstreamflanking sequence of at least 10 nucleotides that is not complementaryto human genomic DNA, and (iii) a downstream flanking sequence of atleast 10 nucleotides that is not complementary to human genomic DNA. 75.The kit of claim 74, further comprising: a second oligonucleotide,wherein the second oligonucleotide comprises a head sequence at that iscomplementary to the upstream flanking sequence of (i) and a tailsequence that is complementary to the downstream flanking sequence of(ii).
 76. The kit of claim 75, wherein the second oligonucleotidecomprises a custom sequence between the head and tail sequences, whereinthe custom sequence is not complementary to human genomic DNA.
 77. Thekit of claim 74, wherein the internal target-complementary sequence isin the range of 10 to 50 nucleotides in length.
 78. The kit of claim 74,wherein the upstream and downstream flanking sequences of (ii) and (iii)are independently in the range of 10 to 40 nucleotides in length. 79.The kit of claim 74, wherein the first set of targeting oligonucleotidescomprises at least 5,000 targeting oligonucleotides.
 80. The kit ofclaim 74, further comprising a second set of at least 1,000 targetingoligonucleotides, each comprising: (i) an internal target-complementarysequence that is in the range of 10 to 100 nucleotides in length andcomplementary to a sequence in the human genome that is not in humanchromosome 21, wherein the different members of the second set havedifferent internal target-complementary sequences (ii) the upstreamflanking sequence, and (iii) the downstream flanking sequence.
 81. Thekit of claim 80, wherein the internal target-complementary sequences ofthe second set are complementary to a sequences in human chromosome 13or 18.